SummaryThe HPr kinase of Gram-positive bacteria is an ATPdependent serine protein kinase, which phosphorylates the HPr protein of the bacterial phosphotransferase system (PTS) and is involved in the regulation of carbohydrate metabolism. The hprK gene from Enterococcus faecalis was cloned via polymerase chain reaction (PCR) and sequenced. The deduced amino acid sequence was confirmed by microscale Edman degradation and mass spectrometry combined with collision-induced dissociation of tryptic peptides derived from the HPr kinase of E. faecalis. The gene was overexpressed in Escherichia coli, which does not contain any ATP-dependent HPr kinase or phosphatase activity. The homogeneous recombinant protein exhibits the expected HPr kinase activity as well as a P-SerHPr phosphatase activity, which was assumed to be a separate enzyme activity. The bifunctional HPr kinase/ phosphatase acts preferentially as a kinase at high ATP levels of 2 mM occurring in glucose-metabolizing Streptococci. At low ATP levels, the enzyme hydrolyses P-Ser-HPr. In addition, high concentrations of phosphate present under starvation conditions inhibit the HPr kinase activity. Thus, a putative function of the enzyme may be to adjust the ratio of HPr and P-Ser-HPr according to the metabolic state of the cell; P-Ser-HPr is involved in carbon catabolite repression and regulates sugar uptake via the phosphotransferase system (PTS). Reinvestigation of the previously described Bacillus subtilis HPr kinase revealed that it also possesses P-Ser-HPr phosphatase activity. However, contrary to the E. faecalis enzyme, ATP alone was not sufficient to switch the phosphatase activity of the B. subtilis enzyme to the kinase activity. A change in activity of the B. subtilis HPr kinase was only observed when fructose-1,6-bisphosphate was also present.
The oligomeric bifunctional HPr kinase/P‐Ser‐HPr phosphatase (HprK/P) regulates many metabolic functions in Gram‐positive bacteria by phosphorylating the phosphocarrier protein HPr at Ser46. We isolated Lactobacillus casei hprK alleles encoding mutant HprK/Ps exhibiting strongly reduced phosphatase, but almost normal kinase activity. Two mutations affected the Walker motif A of HprK/P and four a conserved C‐terminal region in contact with the ATP‐binding site of an adjacent subunit in the hexamer. Kinase and phosphatase activity appeared to be closely associated and linked to the Walker motif A, but dephosphorylation of seryl‐phosphorylated HPr (P‐Ser‐HPr) is not simply a reversal of the kinase reaction. When the hprKV267F allele was expressed in Bacillus subtilis, the strongly reduced phosphatase activity of the mutant enzyme led to increased amounts of P‐Ser‐HPr. The hprK V267F mutant was unable to grow on carbohydrates transported by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and on most non‐PTS carbohydrates. Disrupting ccpA relieved the growth defect only on non‐PTS sugars, whereas replacing Ser46 in HPr with alanine also restored growth on PTS substrates.
Catabolite repression of a number of catabolic operons in bacilli is mediated by the catabolite control protein CcpA, the phosphocarrier protein HPr from the phosphoenolpyruvate-dependent sugar transport system (PTS), and a cis-acting DNA sequence termed the catabolite-responsive element (cre). We present evidence that CcpA interacts with HPr that is phosphorylated at Ser 46 (Ser(P) HPr) and that these proteins form a specific ternary complex with cre DNA. Titration experiments following the circular dichroism signal of the cre DNA indicate that this complex consists of two molecules of Ser(P) HPr, a CcpA dimer, and the cre sequence. Limited proteolysis experiments indicate that the domain structure of CcpA is similar to other members of the LacI/GalR family of helix-turn-helix proteins, comprised of a helix-turn-helix DNA domain and a C-terminal effector domain. NMR titration of Ser(P) HPr demonstrates that the isolated C-terminal domain of CcpA forms a specific complex with Ser(P) HPr but not with unphosphorylated HPr. Based upon perturbations to the NMR spectrum, we propose that the binding site of the C-terminal domain of CcpA on Ser(P) HPr forms a contiguous surface that encompasses both Ser(P) 46 and His 15 , the site of phosphorylation by enzyme I of the PTS. This allows CcpA to recognize the phosphorylation state of HPr, effectively linking the process of sugar import via the PTS to catabolite repression in bacilli. Catabolite repression (CR)1 in Escherichia coli has provided a general paradigm for understanding the regulation of the synthesis of various catabolic enzymes in response to the availability of rapidly metabolizable carbon sources. The mechanism for CR in bacteria such as E. coli involves the cyclic AMP-dependent action of the catabolite gene activator protein, CAP (1). In Bacillus subtilis, however, the mechanism is completely different, because no cyclic AMP or CAP homologue is present (2). CR in some bacilli and a few other Gram-positive organisms has been shown to be dependent on the catabolite control protein A (CcpA), which is a member of the LacI/GalR family of regulators (3, 4), and a cis-active operator DNA sequence, termed the catabolite-responsive element (cre), which has been identified in the promoter or in the 5Ј region of 29 B. subtilis genes (5).There is growing evidence that CR is mechanistically linked to the phosphoenolpyruvate-dependent sugar transport system (PTS), which is responsible for the import of various sugars (1). Mutation of Ser 46 to Ala in the PTS phosphocarrier protein HPr results in resistance to CR for several catabolic genes in B. subtilis (6). Ser 46 in HPr is known to be phosphorylated by an ATP-dependent kinase that is activated by glycolytic intermediates such as fructose-1,6-diphosphate (7). Confirming the link between CR and the PTS, recent DNase I protection experiments have shown that cre sequences are specifically protected by CcpA only in the presence of HPr phosphorylated at Ser 46 (Ser(P) HPr) (8). Other mechanisms for CR have been established...
We have cloned and sequenced the Lactobacillus casei ptsH and ptsI genes, which encode enzyme I and HPr, respectively, the general components of the phosphoenolpyruvate–carbohydrate phosphotransferase system (PTS). Northern blot analysis revealed that these two genes are organized in a single‐transcriptional unit whose expression is partially induced. The PTS plays an important role in sugar transport in L. casei, as was confirmed by constructing enzyme I‐deficient L. casei mutants, which were unable to ferment a large number of carbohydrates (fructose, mannose, mannitol, sorbose, sorbitol, amygdaline, arbutine, salicine, cellobiose, lactose, tagatose, trehalose and turanose). Phosphorylation of HPr at Ser‐46 is assumed to be important for the regulation of sugar metabolism in Gram‐positive bacteria. L. casei ptsH mutants were constructed in which phosphorylation of HPr at Ser‐46 was either prevented or diminished (replacement of Ser‐46 of HPr with Ala or Thr respectively). In a third mutant, Ile‐47 of HPr was replaced with a threonine, which was assumed to reduce the affinity of P–Ser–HPr for its target protein CcpA. The ptsH mutants exhibited a less pronounced lag phase during diauxic growth in a mixture of glucose and lactose, two PTS sugars, and diauxie was abolished when cells were cultured in a mixture of glucose and the non‐PTS sugars ribose or maltose. The ptsH mutants synthesizing Ser‐46–Ala or Ile‐47–Thr mutant HPr were partly or completely relieved from carbon catabolite repression (CCR), suggesting that the P–Ser–HPr/CcpA‐mediated mechanism of CCR is common to most low G+C Gram‐positive bacteria. In addition, in the three constructed ptsH mutants, glucose had lost its inhibitory effect on maltose transport, providing for the first time in vivo evidence that P–Ser–HPr participates also in inducer exclusion.
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