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PubMed1996

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.

By using both metabolizable and nonmetabolizable sugar substrates of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), we show that PTS sugar uptake into intact cells and membrane vesicles of Lactococcus lactis and Bacillus subtilis is strongly inhibited by high concentrations of any of several metabolizable PTS sugars. Inhibition requires phosphorylation of seryl residue 46 in the phosphocarrier protein of the PTS, HPr, by the metabolite-activated, ATP-dependent protein kinase. Inhibition does not occur when wild-type HPr is replaced by the S46A mutant form of this protein either in vesicles of L. lactis or B. subtilis or in intact cells of B. subtilis. Nonmetabolizable PTS sugar analogs such as 2-deoxyglucose inhibit PTS sugar uptake by a distinct mechanism that is independent of HPr(ser-P) and probably involves cellular phosphoenolpyruvate depletion.

Adenosine Triphosphate↗

PubMed1996

Involvement of the central loop of the lactose permease of Escherichia coli in its allosteric regulation by the glucose-specific enzyme IIA of the phosphoenolpyruvate-dependent phosphotransferase system.

Allosteric regulation of several sugar transport systems such as those specific for lactose, maltose and melibiose in Escherichia coli (inducer exclusion) is mediated by the glucose-specific enzyme IIA (IIAGlc) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Deletion mutations in the cytoplasmic N and C termini of the lactose permease protein, LacY, and replacement of all cysteine residues in LacY with other residues did not prevent IIAGlc-mediated inhibition of lactose uptake, but several point and insertional mutations in the central cytoplasmic loop of this permease abolished transport regulation and IIAGlc binding. The results substantiate the conclusion that regulation of the lactose permease in E. coli by the PTS is mediated by a primary interaction of IIAGlc with the central cytoplasmic loop of the permease.

Allosteric Regulation↗

PubMed1998

Unique regulation of carbohydrate chemotaxis in Bacillus subtilis by the phosphoenolpyruvate-dependent phosphotransferase system and the methyl-accepting chemotaxis protein McpC.

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) plays a major role in the ability of Escherichia coli to migrate toward PTS carbohydrates. The present study establishes that chemotaxis toward PTS substrates in Bacillus subtilis is mediated by the PTS as well as by a methyl-accepting chemotaxis protein (MCP). As for E. coli, a B. subtilis ptsH null mutant is severely deficient in chemotaxis toward most PTS carbohydrates. Tethering analysis revealed that this mutant does respond normally to the stepwise addition of a PTS substrate (positive stimulus) but fails to respond normally to the stepwise removal of such a substrate (negative stimulus). An mcpC null mutant showed no response to the stepwise addition or removal of D-glucose or D-mannitol, both of which are PTS substrates. Therefore, in contrast to E. coli PTS carbohydrate chemotaxis, B. subtilis PTS carbohydrate chemotaxis is mediated by both MCPs and the PTS; the response to positive stimulus is primarily McpC mediated, while the duration or magnitude of the response to negative PTS carbohydrate stimulus is greatly influenced by components of the PTS and McpC. In the case of the PTS substrate D-glucose, the response to negative stimulus is also partially mediated by McpA. Finally, we show that B. subtilis EnzymeI-P has the ability to inhibit B. subtilis CheA autophosphorylation in vitro. We hypothesize that chemotaxis in the spatial gradient of the capillary assay may result from a combination of a transient increase in the intracellular concentration of EnzymeI-P and a decrease in the concentration of carbohydrate-associated McpC as the cell moves down the carbohydrate concentration gradient. Both events appear to contribute to inhibition of CheA activity that increases the tendency of the bacteria to tumble. In the case of D-glucose, a decrease in D-glucose-associated McpA may also contribute to the inhibition of CheA. This bias on the otherwise random walk allows net migration, or chemotaxis, to occur.

Bacillus subtilis↗

PubMed1998

Regulation of the Bacillus subtilis GlcT antiterminator protein by components of the phosphotransferase system.

Bacillus subtilis utilizes glucose as the preferred source of carbon and energy. The sugar is transported into the cell by a specific permease of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) encoded by the ptsGHI operon. Expression of this operon is induced by glucose and requires the action of a positive transcription factor, the GlcT antiterminator protein. Glucose availability is sensed by glucose-specific enzyme II (EIIGlc), the product of ptsG. In the absence of inducer, the glucose permease negatively controls the activity of the antiterminator. The GlcT antiterminator has a modular structure. The isolated N-terminal part contains the RNA-binding protein and acts as a constitutively acting antiterminator. GlcT contains two PTS regulation domains (PRDs) at the C terminus. One (PRD-I) is the target of negative control exerted by EIIGlc. A conserved His residue (His-104 in GlcT) is involved in inactivation of GlcT in the absence of glucose. It was previously proposed that PRD-containing transcriptional antiterminators are phosphorylated and concomitantly inactivated in the absence of the substrate by their corresponding PTS permeases. The results obtained with B. subtilis glucose permease with site-specific mutations suggest, however, that the permease might modulate the phosphorylation reaction without being the phosphate donor.

Amino Acid Sequence↗

PubMed1998

Modulation of Escherichia coli adenylyl cyclase activity by catalytic-site mutants of protein IIA(Glc) of the phosphoenolpyruvate:sugar phosphotransferase system.

It is demonstrated here that in Escherichia coli, the phosphorylated form of the glucose-specific phosphocarrier protein IIA(Glc) of the phosphoenolpyruvate:sugar phosphotransferase system is an activator of adenylyl cyclase and that unphosphorylated IIA(Glc) has no effect on the basal activity of adenylyl cyclase. To elucidate the specific role of IIA(Glc) phosphorylation in the regulation of adenylyl cyclase activity, both the phosphorylatable histidine (H90) and the interactive histidine (H75) of IIA(Glc) were mutated by site-directed mutagenesis to glutamine and glutamate. Wild-type IIA(Glc) and the H75Q mutant, in which the histidine in position 75 has been replaced by glutamine, were phosphorylated by the phosphohistidine-containing phosphocarrier protein (HPr-P) and were equally potent activators of adenylyl cyclase. Neither the H90Q nor the H90E mutant of IIA(Glc) was phosphorylated by HPr-P, and both failed to activate adenylyl cyclase. Furthermore, replacement of H75 by glutamate inhibited the appearance of a steady-state level of phosphorylation of H90 of this mutant protein by HPr-P, yet the H75E mutant of IIA(Glc) was a partial activator of adenylyl cyclase. The H75E H90A double mutant, which cannot be phosphorylated, did not activate adenylyl cyclase. This suggests that the H75E mutant was transiently phosphorylated by HPr-P but the steady-state level of the phosphorylated form of the mutant protein was decreased due to the repulsive forces of the negatively charged glutamate at position 75 in the catalytic pocket. These results are discussed in the context of the proximity of H75 and H90 in the IIA(Glc) structure and the disposition of the negative charge in the modeled glutamate mutants.

Adenylyl Cyclases↗

PubMed1999

Lactobacillus casei 64H contains a phosphoenolpyruvate-dependent phosphotransferase system for uptake of galactose, as confirmed by analysis of ptsH and different gal mutants.

Galactose metabolism in Lactobacillus casei 64H was analyzed by genetic and biochemical methods. Mutants with defects in ptsH, galK, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin. ptsH mutations abolish growth on lactose, cellobiose, N-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, in galK mutants. A mutation in galK in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a galK mutation in combination with a mutation in ptsH completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different.

Bacterial Proteins↗

PubMed1999

Transport of D-xylose in Lactobacillus pentosus, Lactobacillus casei, and Lactobacillus plantarum: evidence for a mechanism of facilitated diffusion via the phosphoenolpyruvate:mannose phosphotransferase system.

We have identified and characterized the D-xylose transport system of Lactobacillus pentosus. Uptake of D-xylose was not driven by the proton motive force generated by malolactic fermentation and required D-xylose metabolism. The kinetics of D-xylose transport were indicative of a low-affinity facilitated-diffusion system with an apparent K(m) of 8.5 mM and a V(max) of 23 nmol min(-1) mg of dry weight(-1). In two mutants of L. pentosus defective in the phosphoenolpyruvate:mannose phosphotransferase system, growth on D-xylose was absent due to the lack of D-xylose transport. However, transport of the pentose was not totally abolished in a third mutant, which could be complemented after expression of the L. curvatus manB gene encoding the cytoplasmic EIIB(Man) component of the EII(Man) complex. The EII(Man) complex is also involved in D-xylose transport in L. casei ATCC 393 and L. plantarum 80. These two species could transport and metabolize D-xylose after transformation with plasmids which expressed the D-xylose-catabolizing genes of L. pentosus, xylAB. L. casei and L. plantarum mutants resistant to 2-deoxy-D-glucose were defective in EII(Man) activity and were unable to transport D-xylose when transformed with plasmids containing the xylAB genes. Finally, transport of D-xylose was found to be the rate-limiting step in the growth of L. pentosus and of L. plantarum and L. casei ATCC 393 containing plasmids coding for the D-xylose-catabolic enzymes, since the doubling time of these bacteria on D-xylose was proportional to the level of EII(Man) activity.

Adenosine Triphosphate↗

PubMed2000

Regulation of transcription of the mph(A) gene for macrolide 2'-phosphotransferase I in Escherichia coli: characterization of the regulatory gene mphR(A).

The synthesis of macrolide 2'-phosphotransferase I [Mph(A)], which inactivates erythromycin, is inducible by erythromycin. The expression of high-level resistance to erythromycin requires the mph(A) and mrx genes, which encode Mph(A) and an unidentified protein, respectively. We have studied the mphR(A) gene, which regulates the inducible expression of mph(A). An analysis of the synthesis of Mph(A) in minicells and results of a complementation test indicated that mphR(A) is located downstream from mrx and that its product, MphR(A), represses the production of Mph(A). DNA sequencing indicated that the mph(A), mrx, and mphR(A) genes exist as a cluster that begins with mph(A) and that the deduced amino acid sequence of MphR(A) can adopt an alpha-helix-turn-alpha-helix structure. To study the regulation of gene expression by MphR(A), we performed Northern blotting and primer extension. A transcript of 2. 9 kb that corresponded to the transcript of mph(A) through mphR(A) was detected, and its level was elevated upon exposure of cells to erythromycin. Gel mobility shift assays and DNase I footprinting indicated that MphR(A) binds specifically to the promoter region of mph(A), and the amount of DNA shifted as a results of the binding of MphR(A) decreased as the concentration of erythromycin was increased. These results indicate that transcription of the mph(A)-mrx-mphR(A) operon is negatively regulated by the binding of a repressor protein, MphR(A), to the promoter of the mph(A) gene and is activated upon inhibition of binding of MphR(A) to the promoter in the presence of erythromycin.

Amino Acid Sequence↗

PubMed2001

Diversity of Streptococcus salivarius ptsH mutants that can be isolated in the presence of 2-deoxyglucose and galactose and characterization of two mutants synthesizing reduced levels of HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase system.

In streptococci, HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS), undergoes multiple posttranslational chemical modifications resulting in the formation of HPr(His approximately P), HPr(Ser-P), and HPr(Ser-P)(His approximately P), whose cellular concentrations vary with growth conditions. Distinct physiological functions are associated with specific forms of HPr. We do not know, however, the cellular thresholds below which these forms become unable to fulfill their functions and to what extent modifications in the cellular concentrations of the different forms of HPr modify cellular physiology. In this study, we present a glimpse of the diversity of Streptococcus salivarius ptsH mutants that can be isolated by positive selection on a solid medium containing 2-deoxyglucose and galactose and identify 13 amino acids that are essential for HPr to properly accomplish its physiological functions. We also report the characterization of two S. salivarius mutants that produced approximately two- and threefoldless HPr and enzyme I (EI) respectively. The data indicated that (i) a reduction in the synthesis of HPr due to a mutation in the Shine-Dalgarno sequence of ptsH reduced ptsI expression; (ii) a threefold reduction in EI and HPr cellular levels did not affect PTS transport capacity; (iii) a twofold reduction in HPr synthesis was sufficient to reduce the rate at which cells metabolized PTS sugars, increase generation times on PTS sugars and to a lesser extent on non-PTS sugars, and impede the exclusion of non-PTS sugars by PTS sugars; (iv) a threefold reduction in HPr synthesis caused a strong derepression of the genes coding for alpha-galactosidase, beta-galactosidase, and galactokinase when the cells were grown at the expense of a PTS sugar but did not affect the synthesis of alpha-galactosidase when cells were grown at the expense of lactose, a noninducing non-PTS sugar; and (v) no correlation was found between the magnitude of enzyme derepression and the cellular levels of HPr(Ser-P).

Bacterial Proteins↗

PubMed2001

Negative control of rpoS expression by phosphoenolpyruvate: carbohydrate phosphotransferase system in Escherichia coli.

The sigma(S) (or sigma(38)) subunit of RNA polymerase, encoded by the rpoS gene, is a crucial regulator in the transcriptional control of a set of genes under stressful conditions, such as nutrient starvation. The expression of rpoS is regulated in a complex manner at the levels of transcription, translation, and stability of the product. Although a number of factors involved in the regulation of rpoS expression have been identified, the underlying molecular mechanisms are not fully understood. In this study, we identified the Crr (or EIIA(Glc)) protein as a novel factor that plays an important role not only in the transcriptional control but also in the translational control of rpoS expression. Crr is an important component in glucose uptake through the well-characterized phosphoenolpyruvate:carbohydrate phosphotransferase system. The results of a series of genetic analyses revealed that Crr negatively controls rpoS translation and transcription. The observed transcriptional control by Crr appears to be mediated by cyclic AMP. However, it was found that Crr negatively controls rpoS translation rather directly. These results suggest a possible linkage between the control of rpoS expression and carbon metabolism.

Bacterial Proteins↗

PubMed2003

Involvement of an inducible fructose phosphotransferase operon in Streptococcus gordonii biofilm formation.

Oral streptococci, such as Streptococcus gordonii, are the predominant early colonizers that initiate biofilm formation on tooth surfaces. Investigation of an S. gordonii::Tn917-lac biofilm-defective mutant isolated by using an in vitro biofilm formation assay showed that the transposon insertion is near the 3' end of an open reading frame (ORF) encoding a protein homologous to Streptococcus mutans FruK. Three genes, fruR, fruK, and fruI, were predicted to encode polypeptides that are part of the fructose phosphotransferase system (PTS) in S. gordonii. These proteins, FruR, FruK, and FruI, are homologous to proteins encoded by the inducible fruRKI operon of S. mutans. In S. mutans, FruR is a transcriptional repressor, FruK is a fructose-1-phosphate kinase, and FruI is the fructose-specific enzyme II (fructose permease) of the phosphoenolpyruvate-dependent sugar PTS. Reverse transcription-PCR confirmed that fruR, fruK, and fruI are cotranscribed as an operon in S. gordonii, and the transposon insertion in S. gordonii fruK::Tn917-lac resulted in a nonpolar mutation. Nonpolar inactivation of either fruK or fruI generated by allelic replacement resulted in a biofilm-defective phenotype, whereas a nonpolar mutant with an inactivated fruR gene retained the ability to form a biofilm. Expression of fruK, as measured by the beta-galactosidase activity of the fruK::Tn917-lac mutant, was observed to be growth phase dependent and was enhanced when the mutant was grown in media with high levels of fructose, sucrose, xylitol, and human serum, indicating that the fructose PTS operon was fructose and xylitol inducible, similar to the S. mutans fructose PTS. The induction by fructose was inhibited by the presence of glucose, indicating that glucose is able to catabolite repress fruK expression. Nonpolar inactivation of the fruR gene in the fruK::Tn917-lac mutant resulted in a greater increase in beta-galactosidase activity when the organism was grown in media supplemented with fructose, confirming that fruR is a transcriptional repressor of the fructose PTS operon. These results suggest that the regulation of fructose transport and metabolism in S. gordonii is intricately tied to carbon catabolite control and the ability to form biofilms. Carbon catabolite control, which modulates carbon flux in response to environmental nutritional levels, appears to be important in the regulation of bacterial biofilms.

Base Sequence↗

PubMed2003

In vivo analysis of HPr reveals a fructose-specific phosphotransferase system that confers high-affinity uptake in Streptomyces coelicolor.

HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS), serves multiple functions in carbohydrate uptake and carbon source regulation in low-G+C-content gram-positive bacteria and in gram-negative bacteria. To assess the role of HPr in the high-G+C-content gram-positive organism Streptomyces coelicolor, the encoding gene, ptsH, was deleted. The ptsH mutant BAP1 was impaired in fructose utilization, while growth on other carbon sources was not affected. Uptake assays revealed that BAP1 could not transport appreciable amounts of fructose, while the wild type showed inducible high-affinity fructose transport with an apparent K(m) of 2 microM. Complementation and reconstitution experiments demonstrated that HPr is indispensable for a fructose-specific PTS activity. Investigation of the putative fruKA gene locus led to identification of the fructose-specific enzyme II permease encoded by the fruA gene. Synthesis of HPr was not specifically enhanced in fructose-grown cells and occurred also in the presence of non-PTS carbon sources. Transcriptional analysis of ptsH revealed two promoters that are carbon source regulated. In contrast to what happens in other bacteria, glucose repression of glycerol kinase was still operative in a ptsH background, which suggests that HPr is not involved in general carbon regulation. However, fructose repression of glycerol kinase was lost in BAP1, indicating that the fructose-PTS is required for transduction of the signal. This study provides the first molecular genetic evidence of a physiological role of the PTS in S. coelicolor.

Amino Acid Sequence↗

PubMed2003

Uptake of N,N'-diacetylchitobiose [(GlcNAc)2] via the phosphotransferase system is essential for chitinase production by Serratia marcescens 2170.

The chiR gene of Serratia marcescens 2170, encoding a LysR-type transcriptional activator, was identified previously as an essential factor for expression of chitinases and a chitin-binding protein, CBP21. To identify other genes that are essential for chitinase production, transposon mutagenesis with mini-Tn5Km1 was carried out, and 25 mutants that were unable to produce chitinases and CBP21 were obtained. Analysis of the mutated gene of one of the mutants, N22, revealed the presence of a pts operon in this bacterium, and a mutation was found in ptsI in the operon. In addition to its inability to produce chitinase, N22 did not grow well on N-acetyl-D-glucosamine (GlcNAc), (GlcNAc)(2), and some other carbon sources, most of which were phosphotransferase system (PTS) sugars. Thus, the inability to produce chitinase was assumed to be caused by the defect in uptake of (GlcNAc)(2) via the PTS, considering that (GlcNAc)(2) is the minimal substrate for chitinase induction and the major product of chitin hydrolysis by chitinases of this bacterium. To confirm this assumption, the chb operon, encoding the (GlcNAc)(2)-specific enzyme II permease, was cloned by reference to its Escherichia coli counterpart, and the Serratia chb operon was shown to comprise chbB, chbC, bglA, chbR, and chbG. Disruption of chbC drastically reduced production of chitinases and CBP21 and impaired growth on colloidal chitin. These results indicate that uptake of (GlcNAc)(2) is mediated by the PTS and that the (GlcNAc)(2)-specific enzyme II permease constitutes its major pathway. Since (GlcNAc)(2) uptake is essential for induction of chitinases and CBP21 production, (GlcNAc)(2) appears to be the key molecule in recognition and utilization of chitin by S. marcescens.

Chitin↗

PubMed2004

The Lactobacillus casei ptsHI47T mutation causes overexpression of a LevR-regulated but RpoN-independent operon encoding a mannose class phosphotransferase system.

A proteome analysis of Lactobacillus casei mutants that are affected in carbon catabolite repression revealed that a 15-kDa protein was strongly overproduced in a ptsHI47T mutant. This protein was identified as EIIA of a mannose class phosphotransferase system (PTS). A 7.1-kb DNA fragment containing the EIIA-encoding open reading frame and five other genes was sequenced. The first gene encodes a protein resembling the RpoN (sigma54)-dependent Bacillus subtilis transcription activator LevR. The following pentacistronic operon is oriented in the opposite direction and encodes four proteins with strong similarity to the proteins of the B. subtilis Lev-PTS and one protein of unknown function. The genes present on the 7.1-kb DNA fragment were therefore called levR and levABCDX. The levABCDX operon was induced by fructose and mannose. No "-12, -24" promoter typical of RpoN-dependent genes precedes the L. casei lev operon, and its expression was therefore RpoN independent but required LevR. Phosphorylation of LevR by P approximately His-HPr stimulates its activity, while phosphorylation by P approximately EIIBLev inhibits it. Disruption of the EIIBLev-encoding levB gene therefore led to strong constitutive expression of the lev operon, which was weaker in a strain carrying a ptsI mutation preventing phosphorylation by both P approximately EIIBLev and P approximately His-HPr. Expression of the L. casei lev operon is also subject to P-Ser-HPr-mediated catabolite repression. The observed slow phosphoenolpyruvate- and ATP-dependent phosphorylation of HPrI47T as well as the slow phosphoryl group transfer from the mutant P approximately His-HPr to EIIALev are assumed to be responsible for the elevated expression of the lev operon in the ptsHI47T mutant.

Amino Acid Sequence↗

PubMed2004

Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A.

Polyphosphate:AMP phosphotransferase (PAP) has been identified as an enzyme that catalyzes the phosphorylation of AMP with inorganic polyphosphates [poly(P)] as phosphate donors. We found that the purified PAP of Acinetobacter johnsonii 210A has poly(P) synthetic activity. The PAP catalyzes the dephosphorylation of ADP and processively synthesizes poly(P) of 200 to 700 residues. Comparatively lower concentrations of MgCl(2) (20 mM) were required to obtain optimum poly(P) synthetic activity, whereas higher concentrations of MgCl(2) (100 mM) were necessary for optimum PAP activity. ADP is preferred over GDP as a phosphate donor for poly(P) synthesis. The K(m) and V(max) values for ADP in the poly(P) synthetic activity of PAP were 8.3 mM and 55 micromol min(-1) mg(-1), respectively. We concluded that the PAP of A. johnsonii 210A is a novel type of poly(P) kinase that uses ADP and GDP as substrates.

Acinetobacter↗

PubMed2004

The sorbitol phosphotransferase system is responsible for transport of 2-C-methyl-D-erythritol into Salmonella enterica serovar typhimurium.

2-C-methyl-D-erythritol 4-phosphate is the first committed intermediate in the biosynthesis of the isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate. Supplementation of the growth medium with 2-C-methyl-D-erythritol has been shown to complement disruptions in the Escherichia coli gene for 1-deoxy-D-xylulose 5-phosphate synthase, the enzyme that synthesizes the immediate precursor of 2-C-methyl-D-erythritol 4-phosphate. In order to be utilized in isoprenoid biosynthesis, 2-C-methyl-D-erythritol must be phosphorylated. We describe the construction of Salmonella enterica serovar Typhimurium strain RMC26, in which the essential gene encoding 1-deoxy-D-xylulose 5-phosphate synthase has been disrupted by insertion of a synthetic mevalonate operon consisting of the yeast ERG8, ERG12, and ERG19 genes, responsible for converting mevalonate to isopentenyl diphosphate under the control of an arabinose-inducible promoter. Random mutagenesis of RMC26 produced defects in the sorbitol phosphotransferase system that prevented the transport of 2-C-methyl-D-erythritol into the cell. RMC26 and mutant strains of RMC26 unable to grow on 2-C-methyl-D-erythritol were incubated in buffer containing mevalonate and deuterium-labeled 2-C-methyl-D-erythritol. Ubiquinone-8 was isolated from these cells and analyzed for deuterium content. Efficient incorporation of deuterium was observed for RMC26. However, there was no evidence of deuterium incorporation into the isoprenoid side chain of ubiquinone Q8 in the RMC26 mutants.

Base Sequence↗

PubMed2004

In vivo activity of enzymatic and regulatory components of the phosphoenolpyruvate:sugar phosphotransferase system in Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a pathogenic bacterium that is highly adapted to life on mucosal surfaces. This adaptation is reflected by the very compact genome and the small number of regulatory proteins. However, M. pneumoniae possesses the HPr kinase/phosphorylase (HPrK/P), the key regulator of carbon metabolism in the Firmicutes. In contrast to the enzymes of other bacteria, the HPrK/P of M. pneumoniae is already active at very low ATP concentrations, suggesting a different mode of regulation. In this work, we studied the ability of M. pneumoniae to utilize different carbohydrates and their effects on the activity of the different phosphotransferase system (PTS) components. Glucose served as the best carbon source, with a generation time of about 30 h. Fructose and glycerol were also used but at lower rates and with lower yields. In contrast, M. pneumoniae is unable to use mannitol even though the bacterium is apparently equipped with all the genes required for mannitol catabolism. This observation is probably a reflection of the continuing and ongoing reduction of the M. pneumoniae genome. The general enzymatic and regulatory components of the PTS, i.e., enzyme I, HPr, and HPrK/P, were present under all growth conditions tested in this study. However, HPrK/P activity is strongly increased if the medium contains glycerol. Thus, the control of HPrK/P in vivo differs strongly between M. pneumoniae and the other Firmicutes. This difference may relate to the specific conditions on lipid-rich cell surfaces.

Bacterial Proteins↗

PubMed2004

Characterization of soluble enzyme II complexes of the Escherichia coli phosphotransferase system.

Plasmid-encoded His-tagged glucose permease of Escherichia coli, the enzyme IIBCGlc (IIGlc), exists in two physical forms, a membrane-integrated oligomeric form and a soluble monomeric form, which separate from each other on a gel filtration column (peaks 1 and 2, respectively). Western blot analyses using anti-His tag monoclonal antibodies revealed that although IIGlc from the two fractions migrated similarly in sodium dodecyl sulfate gels, the two fractions migrated differently on native gels both before and after Triton X-100 treatment. Peak 1 IIGlc migrated much more slowly than peak 2 IIGlc. Both preparations exhibited both phosphoenolpyruvate-dependent sugar phosphorylation activity and sugar phosphate-dependent sugar transphosphorylation activity. The kinetics of the transphosphorylation reaction catalyzed by the two IIGlc fractions were different: peak 1 activity was subject to substrate inhibition, while peak 2 activity was not. Moreover, the pH optima for the phosphoenolpyruvate-dependent activities differed for the two fractions. The results provide direct evidence that the two forms of IIGlc differ with respect to their physical states and their catalytic activities. These general conclusions appear to be applicable to the His-tagged mannose permease of E. coli. Thus, both phosphoenolpyruvate-dependent phosphotransferase system enzymes exist in soluble and membrane-integrated forms that exhibit dissimilar physical and kinetic properties.

Cell Membrane↗