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PubMed1991

A bifunctional fusion between beta-glucuronidase and neomycin phosphotransferase: a broad-spectrum marker enzyme for plants.

We have used an in vivo selection approach to isolate a gene encoding a bifunctional fusion peptide between Escherichia coli beta-glucuronidase (GUS) and neomycin phosphotransferase II (NPT-II) from transposon Tn5 in the NH2-GUS::NPT-II-COOH configuration. The fused gene is predicted to encode a fusion peptide 885 amino acids long, and was shown in E. coli to synthesize a 97-kDa GUS+ NPT-II+ gene product. Gel-filtration chromatography suggested that, while the native GUS may be active as a dimer and NPT-II as a monomer, the elution profile of the fusion protein is consistent with that of a trimer. The fusion marker has been produced and defined in transgenic Nicotiana tabacum plants, where both the chimeric gene and the gene product were stable. The bifunctional gene enabled direct KmR selection at the callus stage and enzymatic or histochemical assessment of the steady-state production of GUS activity in regenerated plants. In addition to allowing structure-function determination for the GUS and NPT-II domains of the fusion peptide, the gus::npt-II gene simplifies vector constructs where both marker domains are desired.

Amino Acid Sequence↗

PubMed1992

Cloning, sequencing and expression in Escherichia coli of the ptsI gene encoding enzyme I of the phosphoenolpyruvate:sugar phosphotransferase transport system from Streptococcus salivarius.

We present the cloning and sequencing of the ptsI gene, encoding enzyme I (EI) of the phosphoenolpyruvate (PEP): sugar phosphotransferase (PTS) transport system from Streptococcus salivarius. The ptsI gene corresponds to an open reading frame of 1731 nucleotides, which translates into a putative 577-amino acid (aa) protein with a M(r) of 62,948 and a pI of 4.49. The EI was produced in Escherichia coli under the control of its own promoter located immediately upstream of ptsI, a situation never previously reported for any other gene coding for an EI. The deduced aa sequence of the S. salivarius EI shows a high degree of similarity with the E. coli EI and the EI moiety of the multiphosphoryl transfer protein from Rhodobacter capsulatus. The S. salivarius EI also shares a highly conserved aa cluster with a non-PTS protein, the maize pyruvate:orthophosphate dikinase. The conserved cluster is located in a domain which is hypothesized to be the PEP-binding site.

Amino Acid Sequence↗

PubMedDate not supplied

Diversity of the phosphoenolpyruvate/glucose phosphotransferase system in the Enterobacteriaceae.

The presence of the phosphoenolpyruvate glucose phosphotransferase entry routes was studied in 97 genospecies of Enterobacteriaceae. Phosphoenolpyruvate(PEP)-dependent phosphorylation of alpha-methyl-D-glucoside and 2-deoxyglucose was evidenced in 72 species (group I organisms), suggesting the presence of both the IIGlc (formerly II-BGlc/IIIGlc) and IIMan (formerly II-B/II-AMan) entry routes. Erwinia amylovora, Budvicia aquatica and all species of Leminorella and Proteus (as defined by DNA relatedness studies) showed little or no PEP phosphorylation of 2-deoxyglucose, suggesting the absence of the IIMan entry route (group II organisms). Morganella morganii, Tatumella ptyseos and all species of Xenorhabdus and Providencia (as defined by DNA relatedness studies) showed little or no PEP phosphorylation of alpha-methyl-D-glucoside, suggesting the absence of the IIGlc entry route (group III organisms).

Cell Membrane Permeability↗

PubMedDate not supplied

Involvement of the bacterial phosphotransferase system in diverse mechanisms of transcriptional regulation.

A large number of genes in bacteria appear to be expressed in processes regulated by very different mechanisms dependent on the activities of the proteins of the phosphoenolpyruvate/sugar phosphotransferase system. These mechanisms include protein phosphorylation, antitermination, enhancement, antagonistic repression/activation, sensory detection involving two component systems, and other processes not yet understood.

Bacteria↗

PubMedDate not supplied

Domain shuffling during evolution of the proteins of the bacterial phosphotransferase system.

About twenty permeases of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) have been sequenced and analysed. The results of these analyses suggest that interdomain shuffling, splicing, fusion, deletion, and duplication have occurred repeatedly during their evolution. A uniform nomenclature for these proteins and their domains is proposed.

Biological Evolution↗

PubMedDate not supplied

Mutational analysis of the enzyme IIIGlc of the phosphoenolpyruvate phosphotransferase system in Escherichia coli.

The phosphoenolpyruvate phosphotransferase system (PTS) component EIIIGlc is responsible for transport and phosphorylation of glucose via EIIGlc. It also regulates the catabolism of other carbon sources, such as lactose and maltose, by modulating both the intracellular concentrations of the corresponding inducers and of cAMP. Mutational analysis of EIIIGlc was performed in order to identify crucial residues mediating the interactions between EIIIGlc and its target proteins. Such mutations were isolated by in vitro hydroxylamine mutagenesis of the cloned EIIIGlc gene, crr. Five mutated EIIIGlc impaired in the function of inducer exclusion were obtained. However, these mutations did not abolish the function of EIIIGlc in the transport and phosphorylation of glucose, nor in activation of adenylate cyclase. A single amino acid change was found for each mutation, which is located in a restricted area of the polypeptide chain: Gly47-->Ser47 for the HA2 and HA5 mutations, Ala76-->Thr76 for HA4 mutation and Ser78-->Phe78 for HA3 mutation, indicative of quaternary interactions between the corresponding region of EIIIGlc and its target protein(s).

Adenylyl Cyclases↗

PubMed1992

Interrelationships between protein phosphorylation and oligomerization in transport and chemotaxis via the Escherichia coli mannitol phosphotransferase system.

The membrane-bound enzymes II of the bacterial carbohydrate phosphotransferase system (PTS) are multifunctional: they are required for the transport, phosphorylation and chemotactic sensing of their substrates. An oligomer (minimally a dimer) of at least one of these PTS permeases, the Escherichia coli mannitol permease, appears to be necessary for this protein to optimally carry out these functions. Much indirect evidence is consistent with this hypothesis, and recent experiments show that transport and phosphorylation of, and chemotaxis to, mannitol in E. coli involves an intersubunit phosphotransfer reaction, which can only occur in a protein oligomer. Membrane topological studies of the mannitol permease also argue in favour of an oligomeric structure in the membrane which may be necessary to form the hydrophilic channel through which mannitol must traverse the phospholipid bilayer. The possibility that the oligomerization state of the mannitol permease is a target for regulation of its activity in vivo is proposed, but has not yet been explored experimentally.

Biological Transport, Active↗

PubMed1992

The bacterial phosphotransferase system as a potential vehicle for the entry of novel antibiotics.

During the past twenty-nine years, not a single class of antimicrobial agents has been discovered that has led to new approved human drugs. Despite a dramatic increase in the potency of existing classes, the need for new effective antimicrobial agents continues. The bacterial phosphotransferase system (PTS) offers the possibility of providing new opportunities for the discovery of important agents. This system offers a vehicle for entry into infecting bacteria and pathways for the initiation of metabolism of such agents. Antimicrobial agents which would use the PTS may be found which are active on both growing and sessile bacterial forms, and due to the lack of a eukaryotic PTS counterpart, such analogues may be expected to be non-toxic to the animal host.

Anti-Bacterial Agents↗

PubMedDate not supplied

sigma54-dependent transcription of the Pseudomonas putida xylS operon is influenced by the IIANtr protein of the phosphotransferase system in Escherichia coli.

IIANtr, encoded within the rpoN operons of many Gram-negative bacteria, is a homologue of a class of phosphoryl transfer proteins of the phosphoenolpyruvate: sugar phosphotransferase system. We have used a xylS operon-lacZ fusion from the TOL plasmid of Pseudomonas putida to show that IIANtr influences sigma 54-dependent transcription when the xylS operon is expressed in Escherichia coli. Loss of IIANtr influences, but does not abolish cyclic AMP-independent carbon catabolite repression.

Bacterial Proteins↗

PubMed1990

Evaluation of thymidine kinase and neomycin phosphotransferase II positive selection systems for recovery of genetically atypical and recombinant DNA vaccine viruses.

Dominant phenotypic marker cell culture selection systems were evaluated for the recovery of thymidine kinase positive (TK+), thymidine kinase negative (TK-), and neomycin phosphotransferase II positive (NPT II+) viruses. From 1 to 100 pfu of each marker-positive virus was diluted into 10(6) pfu of marker-negative background virus prior to selection. All three selection systems recovered 100 or fewer pfu of marked virus from the background population (10(-4) fractional level). In some instances, 1 to 10 pfu of marked virus were recovered (10(-5)-10(-6) fractional levels). TK+ and NPT II+ selections yielded nearly pure marker positive virus (98.8% and 99.5% respectively). Populations surviving TK- selection were significantly less pure (58.7%, P less than 0.05). Purity of recovered viruses was independent of the initial fractional level of marker-positive virus. The use of TK+, TK-, and NPT II+ selection systems is discussed in the context of purity testing and environmental surveillance of recombinant DNA viral vaccines.

DNA, Recombinant↗

PubMed1991

High-yield purification of potato tuber pyrophosphate: fructose-6-phosphate 1-phosphotransferase.

The procedure of Yuan et al. (1988, Biochem. Biophys. Res. Commun. 154, 111-117) for the isolation of potato pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) has been modified so that a high yield of homogeneous enzyme could be obtained. Modifications included a lower temperature heat step, a lower percentage initial polyethylene glycol fractionation step (0 to 4%, w/v), stepwise elution following an increase from 30 to 50 mM pyrophosphate during affinity chromatography on Whatman P11 phosphocellulose, anion-exchange chromatography using Q-Sepharose "Fast Flow," and gel filtration chromatography with Superose 6 "Prep grade." Our procedure resulted in an overall 42% yield and a final specific activity of 87 mumol fructose 1,6-bisphosphate produced per minute per milligram protein. Rabbit anti-(potato PFP) polyclonal antibodies effectively immunoprecipitated the activity of both the pure enzyme and the enzyme from a crude extract. Western blot analysis demonstrated that the antibodies were monospecific for PFP. A survey of various potato cultivars demonstrated significant differences in PFP activity with respect to fresh weight. This observation should be taken into consideration before any purification of potato PFP is undertaken.

Chromatography, Affinity↗

PubMedDate not supplied

Overproduction and rapid purification of the phosphoenolpyruvate:sugar phosphotransferase system proteins enzyme I, HPr, and Protein IIIGlc of Escherichia coli.

We present methods for the rapid, simple purification of Enzyme I, HPr, and Protein IIIGlc of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS) using plasmids overproducing gene products. The gene for HPr (ptsH) was cloned into the expression vector pKC30. A simple procedure was devised for the purification to homogeneity of this protein from extracts of heat-induced cells containing pKC30/ptsH recombinant clone. The genes for Enzyme I (ptsI) and Protein IIIGlc (crr) were cloned separately into the expression vector pRE1. Rapid purification procedures were developed for the isolation of homogeneous preparations of these two proteins from extracts of heat-induced cells containing pRE1/ptsI and pRE1/crr recombinants. From about 6 g of cells, these procedures yielded 100, 86, and 50 mg of Enzyme I, HPr, and Protein IIIGlc, respectively. The activity of the proteins purified by these methods was comparable to that of the proteins isolated by previously published less efficient procedures.

Base Sequence↗

PubMed2004

Solution structure of the Escherichia coli YojN histidine-phosphotransferase domain and its interaction with cognate phosphoryl receiver domains.

The Rcs signaling system in Escherichia coli controls a variety of physiological functions, including capsule synthesis, cell division and motility. The activity of the central regulator RcsB is modulated by phosphorylation through the sensor kinases YojN and RcsC, with the YojN histidine phosphotransferase (HPt) domain representing the catalytic unit that coordinates the potentially reversible phosphotransfer reaction between the receiver domains of the RcsB and RcsC proteins. Heteronuclear high-resolution NMR spectroscopy was employed to determine the solution structure of the YojN-HPt domain and to map the interaction with its two cognate receiver domains. The solution structure of YojN-HPt exhibits a well-ordered and rigid protein core consisting of the five helices alphaI to alphaV. The helices alphaII to alphaV form a four-helix bundle signature motif common to proteins of similar function, and helix alphaI forms a cap on top of the bundle. The helix alphaII is separated by a proline induced kink into two parts with different orientations and dynamic behavior that is potentially important for complex formation with other proteins. The N-terminal part of YojN-HPt spanning the first 26 amino acid residues seems to contain neither a regular secondary structure nor a stable tertiary structure and is disordered in solution. The identified YojN-HPt recognition sites for the regulator RcsB and for the isolated receiver domain of the RcsC kinase largely overlap in defined regions of the helices alphaII and alphaIII, but show significant differences. Using the residues with the largest chemical shift changes obtained from titration experiments, we observed a dissociation constant of approximately 200microM for YojN-HPt/RcsC-PR and of 40microM for YojN-HPt/RcsB complexes. Our data indicate the presence of a recognition area in close vicinity to the active-site histidine residue of HPt domains as a determinant of specificity in signal-transduction pathways.

Amino Acid Sequence↗

PubMed2005

Visualization of the phosphorylated active site loop of the cytoplasmic B domain of the mannitol transporter II(Mannitol) of the Escherichia coli phosphotransferase system by NMR spectroscopy and residual dipolar couplings.

The solution structure of a stably phosphorylated form of the cytoplasmic B domain of the mannitol-specific transporter (IIB(Mtl)) of the Escherichia coli phosphotransferase system, containing a mutation of the active site Cys384 to Ser, has been solved by NMR. The strategy employed relies principally on backbone residual dipolar couplings recorded in three different alignment media, supplemented by nuclear Overhauser enhancement data and torsion angle restraints related specifically to the active site loop (residues 383-393). As judged from the dipolar coupling data, the remainder of the structure is unchanged upon phosphorylation within the errors of the coordinates of the previously determined solution structure of unphosphorylated wild-type IIB(Mtl). Thus, only the active site loop was refined. Phosphorylation results in a backbone atomic rms shift of approximately 0.7 angstroms in the active site loop. The resulting conformation is less than 0.5 angstroms away from the equivalent P-loop in both the low and high molecular mass eukaryotic tyrosine phosphatases. 3J(NP) coupling constant measurements using quantitative J-correlation spectroscopy provide a direct demonstration of a hydrogen bond between the phosphoryl group and the backbone amide of Ser391 at position i + 7 from phospho-Ser384, with an approximately linear P-O-H(N) bond angle. The structure also reveals additional hydrogen bonding interactions involving the backbone amides of residues at positions i + 4 and i + 5, and the hydroxyl groups of two serine residues at positions i + 6 and i + 7 that stabilize the phosphoryl group.

Binding Sites↗

PubMed2006

Origin of exopolyphosphatase processivity: Fusion of an ASKHA phosphotransferase and a cyclic nucleotide phosphodiesterase homolog.

The Escherichia coli Ppx protein is an exopolyphosphatase that degrades long-chain polyphosphates in a highly processive reaction. It also hydrolyzes the terminal 5' phosphate of the modified nucleotide guanosine 5' triphosphate 3' diphosphate (pppGpp). The structure of Ppx has been determined to 1.9 A resolution by X-ray crystallography. The exopolyphosphatase is an ASKHA (acetate and sugar kinases, Hsp70, actin) phosphotransferase with an active site found in a cleft between the two amino-terminal domains. Analysis of the active site indicates that among the ASKHA phosphotranferases of known structure, Ppx is the closest to the ectonucleoside triphosphate diphosphohydrolases. A third domain forms a six-helix claw that is similar to the catalytic core of the eukaryotic cyclic nucleotide phosphodiesterases. Most of the 29 sulfate ions bound to the Ppx dimer occupy sites where the polyP chain likely binds. An aqueduct that passes through the enzyme provides a physical basis for the enzyme's high processivity.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed2004

Putative mannose-specific phosphotransferase system component IID represses expression of suilysin in serotype 2 Streptococcus suis.

In this study, we generated a genomic mutant library from a North American strain of serotype 2 Streptococcus suis using the pGh9:ISS1 transposition vector. Suilysin is the hemolysin made by S. suis. A hyper-hemolytic mutant was identified by screening for hemolytic phenotype using media with human blood. The hyper-hemolytic phenotype was characterised by a quantitative hemolysis microplate method. The use of green fluorescent protein (GFP) as a reporter also showed that suilysin gene expression was greater in the mutant. DNA sequence analysis of 3.8 kb surrounding the ISS1 insertion site revealed four open reading frames (ORFs) with three consecutive ORFs that belong to a putative mannose-specific phosphotransferase system (PTS). The S. suis gene homologous to mannose permease IID, manN, was interrupted by the transposon. A complementation test showed that manN repressed the expression of suilysin and the absence of manN was responsible for the hyper-hemolytic phenotype. However, both wild type and isogenic hyper-hemolytic mutant S. suis fermented mannose, glucose and lactose. Thus, despite its potential roles in carbohydrate transport, phosphorylation and metabolism, the manN homologue in the putative mannose-specific PTS regulates gene expression in S. suis.

Animals↗

PubMed2005

Adaptation for fast growth on glucose by differential expression of central carbon metabolism and gal regulon genes in an Escherichia coli strain lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system.

Phosphoenolpyruvate (PEP) is a key intermediate of cellular metabolism and a precursor of commercially relevant products. In Escherichia coli 50% of the glucose-derived PEP is consumed by the PEP:carbohydrate phosphotransferase system (PTS) for glucose transport. PTS, encoded by the ptsHIcrr operon, was deleted from JM101 to generate strain PB11 (PTS-Glc-). PB12, a mutant derived from PB11, grows faster than the parental strain on glucose (PTS-Glc+ phenotype). This strain can redirect some of the PEP not utilized by PTS into the high yield synthesis of aromatic compounds from glucose. Here, we report a comparative transcription analysis among these strains of more than 100 genes involved in central carbon metabolism during growth on glucose. It was found that in the PTS- strains that have reduced glucose transport capacities, several genes encoding proteins with functions related to carbon transport and metabolism were upregulated. Therefore, it could be inferred that these strains synthesize autoinducers of these genes when sensing very low internal glucose concentrations, probably for scavenging purposes. This condition that is permanently present in the PTS- strains even when growing in high glucose concentrations allowed the simultaneous utilization of glucose and acetate as carbon sources. It was found that the gal operon is upregulated in these strains, as well as the aceBAK, poxB and acs genes among others. In PB12, glk, pgi, the TCA cycle and certain respiratory genes are also upregulated. A mutation in arcB in PB12 is apparently responsible for the upregulation of the TCA cycle and certain respiratory genes.

Adaptation, Biological↗

PubMed2006

Replacement of the glucose phosphotransferase transport system by galactose permease reduces acetate accumulation and improves process performance of Escherichia coli for recombinant protein production without impairment of growth rate.

Acetate accumulation under aerobic conditions is a common problem in Escherichia coli cultures, as it causes a reduction in both growth rate and recombinant protein productivity. In this study, the effect of replacing the glucose phosphotransferase transport system (PTS) with an alternate glucose transport activity on growth kinetics, acetate accumulation and production of two model recombinant proteins, was determined. Strain VH32 is a W3110 derivative with an inactive PTS. The promoter region of the chromosomal galactose permease gene galP of VH32 was replaced by the strong trc promoter. The resulting strain, VH32GalP+ acquired the capacity to utilize glucose as a carbon source. Strains W3110 and VH32GalP+ were transformed for the production of recombinant TrpLE-proinsulin accumulated as inclusion bodies (W3110-PI and VH32GalP+-PI) and for production of soluble intracellular green fluorescent protein (W3110-pV21 and VH32GalP+-pV21). W3110-pV21 and VH32GalP+-pV21 were grown in batch cultures. Maximum recombinant protein concentration, as determined from fluorescence, was almost four-fold higher in VH32GalP+-pV21, relative to W3110-pV21. Maximum acetate concentration reached 2.8 g/L for W3110-pV21 cultures, whereas a maximum of 0.39 g/L accumulated in VH32GalP+-pV21. W3110-PI and VH32GalP+-PI were grown in batch and fed-batch cultures. Compared to W3110-PI, the engineered strain maintained similar production and growth rate capabilities while reducing acetate accumulation. Specific glucose consumption rate was lower and product yield on glucose was higher in VH32GalP+-PI fed-batch cultures. Altogether, strains with the engineered glucose uptake system showed improved process performance parameters for recombinant protein production over the wild-type strain.

Acetates↗