Expression of the Xylulose 5-Phosphate Phosphoketolase Gene, xpkA , from Lactobacillus pentosus MD363 Is Induced by Sugars That Are Fermented via the Phosphoketolase Pathway and Is Repressed by Glucose Mediated by CcpA and the Mannose Phosphoenolpyruvate Phosphotransferase System

Abstract

Purification of xylulose 5-phosphate phosphoketolase (XpkA), the central enzyme of the phosphoketolase pathway (PKP) in lactic acid bacteria, and cloning and sequence analysis of the encoding gene, xpkA, from Lactobacillus pentosus MD363 are described. xpkA encodes a 788-amino-acid protein with a calculated mass of 88,705 Da. Expression of xpkA in Escherichia coli led to an increase in XpkA activity, while an xpkA knockout mutant of L. pentosus lost XpkA activity and was not able to grow on energy sources that are fermented via the PKP, indicating that xpkA encodes an enzyme with phosphoketolase activity. A database search revealed that there are high levels of similarity between XpkA and a phosphoketolase from Bifidobacterium lactis and between XpkA and a (putative) protein present in a number of evolutionarily distantly related organisms (up to 54% identical residues). Expression of xpkA in L. pentosus was induced by sugars that are fermented via the PKP and was repressed by glucose mediated by carbon catabolite protein A (CcpA) and by the mannose phosphoenolpyruvate phosphotransferase system. Most of the residues involved in correct binding of the cofactor thiamine pyrophosphate (TPP) that are conserved in transketolase, pyruvate decarboxylase, and pyruvate oxidase were also conserved at a similar position in XpkA, implying that there is a similar TPP-binding fold in XpkA.Lactic acid bacteria (LAB) are capable of generating energy by homo-or heterofermentative degradation of sugars. During anaerobic growth of obligately homofermentative LAB in the presence of excess substrate, energy sources like glucose are converted into pyruvate via the Embden-Meyerhoff-Parnas pathway, and the pyruvate is further metabolized to lactate. During heterofermentative degradation sugars are metabolized via the phosphoketolase pathway (PKP), which results in equimolar amounts of CO 2 , lactate, and acetate-ethanol. Heterolactic LAB can be divided into obligately heterofermentative species, in which both hexoses and pentoses are fermented via the PKP, and facultatively heterofermentative organisms, which degrade hexoses via the Embden-Meyerhoff-Parnas pathway and pentoses via the PKP.Xylulose 5-phosphate phosphoketolase is the central enzyme of the PKP. In the presence of inorganic phospate this enzyme converts xylulose 5Ј-phosphate (X5P) into glyceraldehyde 3-phosphate and acetylphosphate. Phosphoketolase activity was first detected in heterofermentative lactobacilli (7,9,12,25), but it has also been found in other organisms, such as Acetobacter xylinum (20, 23), yeasts (6, 21), Thiobacillus novel-

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

“…Transcriptome analysis with B. subtilis ccpA, hprK, and ptsH1 mutants revealed that numerous nitrogen and phosphorus metabolic genes are submitted to CCR or CCA (491). CcpA of Lactobacillus pentosus represses the activity of xylulose 5-phosphate phosphoketolase, the central enzyme of the phosphoketolase pathway (674). In Clostridium perfringens, inactivation of ccpA diminishes the expression of the enterotoxinencoding cpe gene during entry into the stationary growth phase (913).…”

The Acetate Switch

“…9C) (302). XPK has been identified in heterofermentative lactobacilli, Acetobacter xylinum, Thiobacillus novellus, Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Fibrobacter intestinalis, and yeasts (350). Note that XPK is unsimilar to the transketolases (TKT) used by many species (including E. coli) for conversion of X5P.…”

“…This arrangement, reminiscent of xsc and pta, suggests that the acetylϳP formed by XFP also may perform dual metabolic functions: anabolism and energy conservation (387). CcpA-dependent carbon catabolite repression inhibits xpkA expression in Lactobacillus pentosus (350), the opposite of its effect on PTA and ACK in B. subtilis (164,353). Since the PTA-ACK pathway in B. subtilis operates in a strictly catabolic role, this regulatory arrangement suggests that XPK and its homolog XFP operate primarily in their anabolic role.…”

“…Note that XPK is unsimilar to the transketolases (TKT) used by many species (including E. coli) for conversion of X5P. XPK utilizes inorganic phosphate instead of phosphorylated aldose, it produces acetylϳP instead of fructose-6-phosphate (F6P), and its sequence only poorly resembles that of TKT (350).…”

Phosphorylation of Streptococcus salivarius Lactose Permease (LacS) by HPr(His∼P) and HPr(Ser-P)(His∼P) and Effects on Growth

“…Since only half of the His 6 -HPr(Ser-P) was transformed into the doubly phosphorylated form under these conditions, we increased the concentration of His 6 -HPr(Ser-P) twofold in the reaction medium to obtain a concentration of HPr(Ser-P)(HisϳP) similar to the concentration of HPr(HisϳP) ( (20). Circumstantial evidence also suggests that these PTS proteins control sugar metabolism by a mechanism that has yet to be characterized (5,26,38). We thus looked at whether these proteins could phosphorylate His 6 -IIA LacS .…”