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PubMed1985

The phosphoenolpyruvate-dependent fructose-specific phosphotransferase system in Rhodopseudomonas sphaeroides. Mechanism for transfer of the phosphoryl group from phosphoenolpyruvate to fructose.

The phosphoenolpyruvate-dependent fructose-specific phosphotransferase system in Rhodopseudomonas sphaeroides is a membrane-bound complex of two enzymes, an integral membrane protein EII and a soluble factor SF. In media of high ionic strength the binding constant of SF to the membranes is 55 nM. Phosphorylation of SF, the first step in the reaction sequence, has no influence on the binding. The second step is the transfer of the phosphoryl group from SF to EII. The physical existence of both phosphorylated SF and EII is demonstrated and it is shown that the phosphoryl group is donated to the next species in the reaction sequence. The molecular mass of SF is 110 kDa. EII is almost completely extracted from the membrane as a dimer. The molecular mass of the monomer is 55 kDa. Both SF and EII possess thiols that are essential for catalysis. The thiol on EII is protected against inactivation by N-ethylmaleimide in the phosphorylated state. Kinetic experiments show that the binding site for fructose on EII is induced by phosphorylation of EII ('ping-pong' mechanism). The affinity constants of the phosphotransferase complex for phosphoenolpyruvate and fructose at infinite concentration of the other substrate are 25 microM and 8 microM, respectively. The fructose phosphorylation rate equation is given as a function of the concentration of the two enzymes and the two substrates.

Autoradiography↗

PubMed1987

Cloning and expression in Escherichia coli of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus.

The Streptomyces hygroscopicus hyg gene encoding a hygromycin B phosphotransferase has been introduced into different sites of both the Escherichia coli plasmid pBR322 and the Escherichia coli-Saccharomyces cerevisiae shuttle vector YRp7. When this gene was inserted into the BamHI site of pBR322 and then cloned in E. coli phosphorylating activity was not detected, indicating that the hyg gene promoter was not functional in this bacterium. However, when the hyg gene was inserted into either the unique PstI site of pBR322 or into each of the two PstI sites of YRp7, phosphotransferase activity was observed. Analysis of the translation products from these constructions by coupled in vitro transcription-translation systems suggested that in all cases transcrition was regulated by a promoter not provided by the inserted hyg gene and that the synthesized polypeptide was identical to that present in S. hygroscopicus.

Base Sequence↗

PubMed1987

Purification and characterization of a hygromycin B phosphotransferase from Streptomyces hygroscopicus.

A hygromycin B phosphotransferase activity from Streptomyces hygroscopicus has been highly purified by ammonium sulphate fractionation followed by affinity column chromatography through Sepharose-6B-hygromycin-B. The combined active fractions showed a single protein band (41 kDa) when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. When gel electrophoresis was performed under non-denaturing conditions, the single protein band promoted in situ phosphorylation of hygromycin B, indicating that this protein corresponded to the purified hygromycin B phosphotransferase. The enzyme has been purified 236-fold and approximate Km values of 0.56 microM for hygromycin B and ATP, respectively, were deduced.

Kanamycin Kinase↗

PubMed1995

Lactose-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. Purification of the histidine-tagged transmembrane component IICBLac and its hydrophilic IIB domain by metal-affinity chromatography, and functional characterization.

The lactose-specific integral-membrane-protein enzyme II (IICBLac) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus catalyses the uptake and phosphorylation of lactose. It consists of an N-terminal membrane-spanning IIC domain and a C-terminal hydrophilic IIB domain. IICBLac was fused with a C-terminal tag of six histidine residues using recombinant DNA technology. The resulting protein, IICBLac-His, was produced in Escherichia coli and purified under nondenaturing conditions to homogenity. The purification procedure consists of a NaOH extraction step followed by solubilisation with Triton X-100, and metal-affinity chromatography using Ni(2+)-nitrilotriacetic acid resin. The purified recombinant His-tagged protein possessed substrate specificity identical to that of the wild-type protein. To investigate the hydrophilic IIB domain, the DNA sequence coding for IIB and the His tag were fused in-frame to a DNA sequence specific for an initiation signal. The overproduced recombinant IIBLac-His was obtained by metal-affinity chromatography in pure form. Bacterial phosphotransferase-system-dependent phosphorylation of IIB-His was demonstrated in a photometric assay and by urea/polyacrylamide gel electrophoresis. The phosphorylation activity of the mutant protein [C476S]-IICBLac, containing the mutagenized phosphorylation site, was restored in the presence of IIBLac-His in a phosphorylation assay.

Base Sequence↗

PubMed1996

Cloning and nucleotide sequence of the mphB gene for macrolide 2'-phosphotransferase II in Escherichia coli.

Macrolide 2'-phosphotransferase II [MPH(2')II] inactivates macrolide antibiotics. The mphB gene for MPH(2')II was cloned from Escherichia coli and sequenced. Analysis of the nucleotide sequence indicated that mphB encoded a protein of 302 amino acids with a molecular mass of 34483 Da. The carboxy terminal region of the deduced protein contained a sequence that resembled a conserved functional domain in aminoglycoside phosphotransferases.

Amino Acid Sequence↗

PubMed1998

Expression of the mphB gene for macrolide 2'-phosphotransferase II from Escherichia coli in Staphylococcus aureus.

The genes mphA and mphB encode macrolide 2'-phosphotransferases I and II, respectively, and they confer resistance to macrolide antibiotics in Escherichia coli. To study the expression of these genes in Gram-positive bacteria, we constructed recombinant plasmids that consisted of an mph gene and the pUB110 vector in Bacillus subtilis. When these plasmids were introduced into Staphylococcus aureus, the mphB gene was active and macrolide 2'-phosphotransferase II was produced. The gene endowed S. aureus with high-level resistance to spiramycin, a macrolide antibiotic with a 16-membered ring. Moreover, transcription of the mphB gene in S. aureus began at the promoter that was active in E. coli.

Escherichia coli↗

PubMed1980

Phosphoenolpyruvate-dependent glucose phosphotransferase activity in Streptococcus mitis ATCC 903.

A constitutive phosphoenolpyruvate (PEP)-dependent glucose phosphotransferase system was found in decryptified cells of Strep. mitis. The system displayed saturation kinetics and the apparent Km value for the non-fermentable analogue 2-deoxyglucose (2DG) was 0.4 mM. Sodium fluoride inhibited the activity when 2-phosphoglycerate was used as the energy source instead of PEP. Intact cells accumulated 2DG and also a derivative that behaved chromatographically like 2DG-6-phosphate. PEP-dependent glucose phosphotransferase activity was also demonstrated in freshly collected plaque.

Adult↗

PubMed1985

Development of two DNA probes for differentiating the structural genes of subclasses I and II of the aminoglycoside-modifying enzyme 3'-aminoglycoside phosphotransferase.

Two DNA probes were developed to screen for the genes encoding 3'-aminoglycoside phosphotransferase activity in gram-negative bacilli. The 3'-I phosphotransferase [APH(3')I] probe was subcloned from Tn903; the APH(3')II probe was subcloned from Tn5. Each probe proved to be specific for genes corresponding to its own APH(3') subclass and did not hybridize with DNA from other classes when tested at high stringency by either Southern hybridization or dot-blot hybridization methods. The APH(3')I probe hybridized to DNA obtained from organisms demonstrating APH(3')I activity as measured by the phosphocellulose paper binding assay (PPBA) as well as to DNA from organisms reported to have both APH(3')I and APH(3')II activity by PPBA. This probe did not hybridize to DNA from organisms showing only APH(3')II activity by PPBA. The APH(3')II probe demonstrated homology with DNA from organisms showing APH(3')II activity by PPBA but not with DNA from organisms showing APH(3')I activity or both APH(3')I and APH(3')II activity by PPBA. We conclude that organisms previously believed to contain both APH(3')I and APH(3')II genes based on PPBA contain in fact only the APH(3')I gene.

Autoradiography↗

PubMed1996

Sequence and characterization of a novel chromosomal aminoglycoside phosphotransferase gene, aph (3')-IIb, in Pseudomonas aeruginosa.

A novel, probably chromosomally encoded, aminoglycoside phosphotransferase gene was cloned on a 2,996-bp PstI fragment from Pseudomonas aeruginosa and designated aph (3')-IIb. It coded for a protein of 268 amino acids that showed 51.7% amino acid identity with APH (3')-II [APH(3') is aminoglycoside-3' phosphotransferase] from Tn5. Two other open reading frames on the cloned fragment showed homology to a signal-transducing system in P. aeruginosa.

Aminoglycosides↗

PubMed1998

The Cys607-->Tyr change in the UL97 phosphotransferase confers ganciclovir resistance to two human cytomegalovirus strains recovered from two immunocompromised patients.

Two ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) strains recovered from an AIDS patient (strain VR4990) and a heart transplant recipient (strain VR5474) showed a Cys607-->Tyr change in the UL97-encoded phosphotransferase. No amino acid substitutions were observed in the viral DNA polymerase. Marker transfer experiments showed marked reduction in GCV phosphorylation and drug susceptibility of the recombinant HCMV strain VR4990rec2-1-1. These results further extend the region of the carboxy-terminal domain of the UL97 phosphotransferase involved in GCV substrate recognition.

AIDS-Related Opportunistic Infections↗

PubMed1988

Effect of nutritional constraints on the biosynthesis of the components of the phosphoenolpyruvate: sugar phosphotransferase system in a fresh isolate of Streptococcus mutans.

A procedure for the purification of enzyme I (EI) and the protein HPr, the general components of the phosphoenolpyruvate:sugar phosphotransferase system, from Streptococcus mutans serotype c is presented. The method was also applied successfully to the purification of EI and HPr from Streptococcus salivarius, Streptococcus sobrinus, and Streptococcus sanguis. Using specific antibodies obtained against the proteins purified from S. mutans DR0001, we determined quantitatively by rocket electrophoresis the cellular levels of EI and HPr in a freshly isolated strain of S. mutans grown under various conditions in continuous culture. The activity of a few specific EIIs was also determined by an in vitro phosphorylation test. Results indicated that maximum EII activities for glucose, mannose, and 2-deoxyglucose were obtained under conditions of glucose limitation, at pH 7.0 and low dilution rate (D = 0.057/h). Increasing the amount of glucose or the dilution rate (D = 0.40/h) or decreasing the pH from 7.0 to 5.5 resulted in a 1.4- to 24-fold decrease in these activities. The EII activity for fructose was not influenced by the growth conditions in the same way as the other EIIs. The fructose EII was highest at pH 5.5 and at high dilution rate under conditions of glucose or nitrogen limitation and was always repressed at pH 7.0 and at low dilution rates. The intracellular levels of EI were also dependent on the growth conditions. The highest concentration (0.65 nmol/mg of protein) was observed in cells grown under glucose limitation at pH 7.0 and high dilution rate, and the lowest concentration (0.12 nmol/mg of protein) was found in cells grown under glucose excess at pH 7.0 and high dilution rate. The other general component of the phosphoenolpyruvate:sugar phosphotransferase system, the protein HPr, was not influenced significantly by varying growth conditions.

Bacterial Proteins↗

PubMed1990

Evidence that a low-affinity sucrose phosphotransferase activity in Streptococcus mutans GS-5 is a high-affinity trehalose uptake system.

High-affinity sucrose uptake in the oral pathogen Streptococcus mutans is mediated by the phosphoenolpyruvate-dependent phosphotransferase system. In this report, we provide evidence that a lower-affinity sucrose phosphotransferase system in S. mutans GS-5, previously described by others, is in fact a high-affinity trehalose uptake system that also recognizes sucrose as a substrate.

Biological Transport, Active↗

PubMed2006

YeeI, a novel protein involved in modulation of the activity of the glucose-phosphotransferase system in Escherichia coli K-12.

The membrane-bound protein EIICB(Glc) encoded by the ptsG gene is the major glucose transporter in Escherichia coli. This protein is part of the phosphoenolpyruvate:glucose-phosphotransferase system, a very important transport and signal transduction system in bacteria. The regulation of ptsG expression is very complex. Among others, two major regulators, the repressor Mlc and the cyclic AMP-cyclic AMP receptor protein activator complex, have been identified. Here we report identification of a novel protein, YeeI, that is involved in the regulation of ptsG by interacting with Mlc. Mutants with reduced activity of the glucose-phosphotransferase system were isolated by transposon mutagenesis. One class of mutations was located in the open reading frame yeeI at 44.1 min on the E. coli K-12 chromosome. The yeeI mutants exhibited increased generation times during growth on glucose, reduced transport of methyl-alpha-d-glucopyranoside, a substrate of EIICB(Glc), reduced induction of a ptsG-lacZ operon fusion, and reduced catabolite repression in lactose/glucose diauxic growth experiments. These observations were the result of decreased ptsG expression and a decrease in the amount of EIICB(Glc). In contrast, overexpression of yeeI resulted in higher expression of ptsG, of a ptsG-lacZ operon fusion, and of the autoregulated dgsA gene. The effect of a yeeI mutation could be suppressed by introducing a dgsA deletion, implying that the two proteins belong to the same signal transduction pathway and that Mlc is epistatic to YeeI. By measuring the surface plasmon resonance, we found that YeeI (proposed gene designation, mtfA) directly interacts with Mlc with high affinity.

Chromosomes, Bacterial↗

PubMed1970

Distribution of the phosphoenolpyruvate: glucose phosphotransferase system in bacteria.

A survey of the occurrence of the phosphoenolpyruvate-dependent glucose phosphotransferase system was carried out in a number of bacteria, representing both gram-positive and gram-negative facultative anaerobic and strictly aerobic types. The system was found to be present in representatives of genera that are characteristically facultative anaerobes, but the system was absent in members of those genera that are strictly aerobic. Thus, although the phosphoenolpyruvate phosphotransferase system is an important system for the transport of sugars in bacteria carrying out anaerobic glycolysis, it plays no role in sugar transport by those organisms having a strictly oxidative physiology. A fundamentally different system, probably not involving phosphorylation during transport, is indicated in this latter group.

Bacteria↗

PubMed1980

Regulation of genes coding for enzyme constituents of the bacterial phosphotransferase system.

Regulation of the synthesis of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system was systematically studied in wild-type and mutant strains of Salmonella typhimurium and Escherichia coli. The results suggest that enzyme I and HPr as well as the glucose-specific and the mannose-specific enzymes II are synthesized by a mechanism which depends on (i) cyclic adenosine monophosphate and its receptor protein; (ii) extracellular inducer; (iii) the sugar-specific enzyme II complex which recognizes the inducing sugar; and (iv) the general energy-coupling proteins of the phosphotransferase system, enzyme I and HPr.

Carbohydrate Metabolism↗

PubMed1982

Phosphoenolpyruvate-dependent phosphotransferase system enzyme III and plasmid-encoded sucrose transport in Escherichia coli K-12.

The phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system enzyme IISCR, specific for and regulated by sucrose, was analyzed in derivatives of Escherichia coli K-12 carrying the sucrose plasmid pUR404. Enzyme IIScr, coded for by gene scrA of the plasmid, depended for its transport and phosphorylation activity directly on the phosphotransferase system enzyme IIIGlc, Scr, coded for by the chromosomal gene crr.

Biological Transport↗

PubMed1983

Involvement of lactose enzyme II of the phosphotransferase system in rapid expulsion of free galactosides from Streptococcus pyogenes.

Streptococcus pyogenes accumulated thiomethyl-beta-galactoside as the 6-phosphate ester due to the action of the phosphoenolpyruvate:lactose phosphotransferase system. Subsequent addition of glucose resulted in rapid efflux of the free galactoside after intracellular dephosphorylation (inducer expulsion). Efflux was shown to occur in the apparent absence of the galactose permease, but was inhibited by substrate analogs of the lactose enzyme II and could not be demonstrated in a mutant of S. lactis ML3 which lacked this enzyme. The results suggest that the enzymes II of the phosphotransferase system can catalyze the rapid efflux of free sugar under appropriate physiological conditions.

Biological Transport↗

PubMed1983

The chromosomal 3',5"-aminoglycoside phosphotransferase in Streptococcus pneumoniae is closely related to its plasmid-coded homologs in Streptococcus faecalis and Staphylococcus aureus.

The apparently chromosomally encoded 3',5"-aminoglycoside phosphotransferase (type III), from the high-level aminoglycoside-resistant Streptococcus pneumoniae BM4200, was compared with homologous enzymes coded for by the plasmids pJH1 and pSH2, originally isolated from Streptococcus faecalis and Staphylococcus aureus, respectively, and also found in a wild strain of S. aureus, BM4600. The enzymes appeared to be indistinguishable, and we conclude that the gene encoding 3',5"-aminoglycoside phosphotransferase (type III) can cross generic barriers within gram-positive cocci.

Enterococcus faecalis↗