Evidence for the presence of heat-stable protein (HPr) and ATP-dependent HPr kinase in heterofermentative lactobacilli lacking phosphoenolpyruvate:glycose phosphotransferase activity.

Abstract

An analysis of the biochemical basis for the lack of phosphoenolpyruvate:glycose phosphotransferase activity in heterofermentative lactobacilli was carried out. Extracts of Lactobaciflus brevis and Lactobacilus buchneri failed to reconstitute phosphotransferase activity of extracts of Staphylococcus aureus mutants impaired in the phosphotransferase system due to the absence of enzyme I, enzyme IILc, or enzyme IIILac activity, suggesting that these lactobacilli lack those phosphotransferase system components. In contrast, complementation tests with an extract of a S. aureus mutant deficient in heat-stable protein (HPr) indicated the presence of HPr activity in heterofermentative lactobacilli. The HPr of L. brevis was purified and shown to have properties similar to those of a typical HPr. In addition, L. brevis possesses an ATP-dependent protein kinase that phosphorylates a serine residue of the endogenous HPr as well as other HPrs of Gram-positive origin. The kinase activity is markedly stimulated by phosphorylated compounds related to sugar metabolism and is negatively modulated by orthophosphate, pyrophosphate, or arsenate and by a low molecular weight endogenous factor. In keeping with the idea of a regulatory role for the phosphorylation of HPr in lactobacilli, a HPr[Ser(P)] phosphatase activity in L. brevis was also demonstrated. On the basis of the rinding of HPr and a system for its reversible covalent modification in an organism devoid of a functional phosphotransferase system we propose that, in lactobacilli, HPr has a role in the regulation of pathways other than the phosphotransferase system.One of the general energy coupling components of the phosphoenolpyruvate (P-ePrv):glycose phosphotransferase system (PTS) is a low molecular weight protein known as HPr (heat-stable protein) that is phosphorylated by P-ePrv and enzyme I on a single histidine residue (1). Recent studies have established that, in Gram-positive bacteria, HPr can also be reversibly phosphorylated on a serine residue by a unique, metabolite-activated, ATP-dependent protein kinase-the phosphorylated HPr is designated HPr[Ser(P)]-and a Pi-dependent HPr[Ser(P)] phosphatase (for reviews see refs. 2 and 3). This reversible modification appears to play a regulatory role in energy and carbon metabolism by modulating the flow of phosphate groups at the level of HPr (3, 4). HPr[Ser(P)] has also been implicated in a vectorial regulatory process, termed inducer expulsion, that effectively displaces intracellularly accumulated sugars and functions in response to variations in energy availability and metabolite concentrations (3,5,6).Heterofermentative lactobacilli, such as Lactobacillus brevis or Lactobacillus buchneri, appear to lack a functional PTS (refs. 7 and 8 and this paper). However, these organisms possess a metabolite-activated vectorial process that displaces intracellularly accumulated galactosides (8). This regulatory mechanism resembles the expulsion process of homofermentative lactic acid bacteria, which possess a PTS and...

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

“…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.…”

Sequence analyses and evolutionary relationships among the energy‐coupling proteins enzyme I and HPr of the bacterial phosphoenolpyruvate: Sugar phosphotransferase system

“…Cells were ruptured in a French pressure cell as previously described . Cell debris was removed by centrifugation, and the crude extracts were assayed for Enzyme I and HPr by complementation employing Staphylococcus aureus ptsl and ptsH mutant crude extracts as described previously (Reizer et al, 1988a. Activity is expressed in nanomoles of glucose phosphate formed per hour per milligram of protein.…”

Identification and characterization of phosphoenolpyruvate: fructose phosphotransferase systems in three Streptomyces species

“…The phosphoenolpyruvate : sugar phosphotransferase system (PTS) is known to function in carbohydrate transport, chemotaxis, and a variety of regulatory capacities in diverse bacteria (Reizer et al, 1988b;Titgemeyer, 1993). More than 20 different carbohydrates are transported via the PTS which exclusively uses phosphoenolpyruvate (PEP) as the phosphoryl donor in a phosphoryl transfer chain that involves the two general energy-coupling proteins, Enzyme I and HPr, as well as the sugar-specific, membrane-bound Enzyme I1 complexes.…”