Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

Abstract

We have cloned and sequenced the Lactobacillus casei hprK gene encoding the bifunctional enzyme HPr kinase/P-Ser-HPr phosphatase (HprK/P). Purified recombinant L. casei HprK/P catalyzes the ATP-dependent phosphorylation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system at the regulatory Ser-46 as well as the dephosphorylation of seryl-phosphorylated HPr (P-SerHPr). The two opposing activities of HprK/P were regulated by fructose-1,6-bisphosphate, which stimulated HPr phosphorylation, and by inorganic phosphate, which stimulated the P-Ser-HPr phosphatase activity. A mutant producing truncated HprK/P was found to be devoid of both HPr kinase and P-Ser-HPr phosphatase activities. When hprK was inactivated, carbon catabolite repression of N-acetylglucosaminidase disappeared, and the lag phase observed during diauxic growth of the wild-type strain on media containing glucose plus either lactose or maltose was strongly diminished. In addition, inducer exclusion exerted by the presence of glucose on maltose transport in the wild-type strain was abolished in the hprK mutant. However, inducer expulsion of methyl ␤-D-thiogalactoside triggered by rapidly metabolizable carbon sources was still operative in ptsH mutants altered at Ser-46 of HPr and the hprK mutant, suggesting that, in contrast to the model proposed for inducer expulsion in gram-positive bacteria, P-Ser-HPr might not be involved in this regulatory process.Bacteria can respond to the presence of abundant carbon sources via a complex network of regulatory processes. Rapidly metabolizable carbohydrates such as glucose inhibit expression of catabolic operons encoding the enzymes necessary for the uptake and metabolism of less-favorable carbon sources (43) and also affect the synthesis of enzymes of central metabolic pathways such as glycolytic enzymes, as has been shown in Lactobacillus casei (31) and Lactococcus lactis (28). In grampositive bacteria, the metabolite-activated HPr kinase/P-SerHPr phosphatase (HprK/P) (2, 13, 22, 38) is the first enzyme of the signal transduction pathway used to control several of these regulatory processes. This bifunctional enzyme catalyzes the phosphorylation of HPr at the regulatory seryl residue 46 (5) as well as the dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr) (22). HPr is a phosphocarrier protein of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). The PTS catalyzes the concomitant uptake and phosphorylation of carbohydrates by forming a protein phosphorylation cascade. HPr becomes intermediately phosphorylated by PEP and enzyme I at the catalytic His-15, and P-HisHPr transfers the phosphoryl group to the sugar-specific enzyme II complexes which transport and phosphorylate their corresponding substrates (35).Carbon catabolite repression (CCR) in gram-positive bacteria was found to be one of the P-Ser-HPr-controlled processes.

Properties and Regulation of the Bifunctional Enzyme HPr Kinase/Phosphatase in Bacillus subtilis

“…Whereas 10 mM FBP partly restored the kinase activity for both proteins, HPr and Crh, 10 mM F2,6BP had only a small effect regarding the phosphorylation of Crh, and no effect of F2,6BP was observed for the phosphorylation of HPr (data not shown). Similar results with FBP and the capacity of restoring the kinase activity in the presence of inorganic phosphate were reported for HPrK/P from Lactobacillus casei (63). A similar experiment was performed to evaluate the possibility to restore the kinase activity in an assay using ATP with 0.1 mM inorganic phosphate in the absence of effector molecules.…”

“…On the contrary, inorganic phosphate, acetyl phosphate, and DL-glyceraldehyde 3-phosphate inhibited the kinase activity of HPrK/P. The inhibitory effect of inorganic phosphate on kinase activity has previously been reported for HPr kinases (46,47,(61)(62)(63). To further evaluate the effect of inorganic phosphate, 1 mM inorganic phosphate was included in a kinase assay with increasing concentrations of FBP/ F2,6BP.…”

“…Of the evaluated glycolytic intermediates, FBP as well as inorganic phosphate are known as an allosteric activator and inhibitor, respectively, not only for the kinase activity of HPrK/P from B. subtilis (11) but also for the enzymes from, for example, L. casei (63), Lactobacillus brevis (62), S. mutans (61), and S. pyogenes (47). Furthermore, F2,6BP was also found to be an allosteric activator for the kinase activity.…”

A novel mode of control of Mycoplasma pneumoniae HPr kinase/phosphatase activity reflects its parasitic lifestyle

“…Kinase activity occurs only if the concentration of ATP is high ( 0n2 mM) and glycolytic intermediates such as FBP are present (Jault et al, 2000). Similarly, the enzymes from other low-GC Gram-positive bacteria are active as a kinase only at high ATP concentrations that indicate a good supply of nutrients (Kravanja et al, 1999 ;Brochu & Vadeboncoeur, 1999 ;Dossonnet et al, 2000 ;Huynh et al, 2000). In contrast, M. pneumoniae HPrK\P exhibits an inverse mode of regulation : this enzyme needs P i for phosphatase activity, whereas the kinase activity occurs at ATP concentrations as low as 1 µM.…”

“…1a). Kinase activity of some HPrK\P proteins, like that of B. subtilis and Lactobacillus casei, is stimulated by glycolytic intermediates such as FBP (Galinier et al, 1998 ;Reizer et al, 1998 ;Jault et al, 2000 ;Dossonnet et al, 2000). We analysed the response of the kinase activity of HPrK\P of M. pneumoniae to FBP in the presence of low ATP concentrations (1, 5 and 10 µM).…”