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.     

Evidence of a glucose proton motive force-dependent permease and a fructose phosphoenolpyruvate:phosphotransferase transport system in Lactobacillus reuteri CRL 1098

Sugar uptake and phosphoenolpyruvate phosphorylation assays have shown that the heterofermentative strain Lactobacillus reuteri CRL 1098, of likely probiotic value, can transport D-fructose through an inducible fructose-specific phosphotransferase system (K(m) 95 microM) and D-glucose mainly through a proton motive force-driven permease. These data open new perspectives for metabolic and regulatory studies in this bacterium.     

Evolutionary origin of aminoglycoside phosphotransferase resistance genes

Summary The protein sequences of seven 3-aminoglycoside phosphotransferases falling into the six identified types and three 6-aminoglycoside phosphotransferases were analyzed to give a rooted phylogenetic tree. This tree supports the origin of these groups of enzymes in an ancestor closely related to the actinomycetes, and that horizontal transfer of the resistance genes occurred, possibly via transposons. The implications for genetic engineering of a novel antibiotic are discussed.     

Separation of four components of the phosphoenolpyruvate: glucose phosphotransferase system in Vibrio parahaemolyticus.

Four classes of Vibrio parahaemolyticus mutants defective in the phosphoenolpyruvate: glucose phosphotransferase system (PTS) are described. They were phenotypically different, and were defective in different PTS components. The components designated tentatively as II, I, III, and H were separated by gel filtration of a wild-type extract. Component II, which was specific for glucose and found in the particulate fraction, is probably membrane-bound, glucose-specific enzyme II. Both components I and H were soluble proteins, and the latter was relatively heat-stable. Component I was required for phosphorylation of glucose, trehalose, fructose, mannose, and mannitol. Component H was also required for phosphorylating all the above sugars except fructose. These and some additional findings strongly suggest that components I and H correspond to enzyme I and HPr, respectively. Component III, a soluble heat-stable protein, may be equivalent to the sugar-specific factor III found in other organisms, although it seems to participate in phosphorylating two sugars, glucose and trehalose. There were evidences that mutants defective in components I and III were deficient in cyclic adenosine 3',5'-monophosphate synthesis under certain conditions.     

Identification of the genes for the lactose-specific components of the phosphotransferase system in the lac operon of Staphylococcus aureus

The nucleotide and deduced amino acid sequences of the lacE and lacF genes, which code for the lactose-specific Enzyme II and Enzyme III of the Staphylococcus aureus phosphotransferase system, are presented. The primary translation products consist of a hydrophobic protein of 572 amino acids (Mr = 62,688) and a polypeptide of 103 amino acids (Mr = 11,372), respectively. The assignment of lacF as the gene for Enzyme IIIlac was based upon the known amino acid sequence of the protein. The identity of lacE as encoding Enzyme IIlac was based upon immunoreactivity of the cloned gene product with antibodies raised against purified Enzyme IIlac from S. aureus and an assay of biological function of the protein expressed in Escherichia coli. The order of the known genes of the S. aureus lac operon is lacF-lacE-lacG, the latter encoding phospho-beta-galactosidase.     

Physiological significance of the concentration of human milk glucose

Summary The concentration of glucose in human milk, [Glucose]_milk, was directly related to the volume of milk secreted not only during lactogenesis and weaning but also during full lactation. To investigate the mechanism for this observation we first established that glucose equilibrates across the apical membrane of the mammary alveolar cell, using infusion of stable isotopically labelled glucose into lactating women. Our results indicate that [Glucose]_milk can be used to measure the glucose concentration in the mammary alveolar cell, [Glucose]_cell. We then investigated the regulation of glucose transport into the mammary alveolar cell using glucose clamp methodology in fully lactating and weaning women. Maintenance of high plasma insulin concentrations for four hours under euglycemic conditions had no effect on [Glucose]_milk, demonstrating that insulin does not regulate glucose transport into the mammary gland. On the other hand, maintaining the [Glucose]_plasma at twice the fasting level resulted in a 3-fold increase in the steady state [Glucose]_milk in fully lactating women and a 5-fold increase in [Glucose]_milk in weaning women. Kinetic analysis of the data showed that the V_max for glucose transport into the mammary alveolar cell across the basolateral membrane is regulated by the level of synthetic activity in the mammary alveolar cell.Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

Determination of 3-deoxy-D-arabino-heptulosonate 7-phosphate productivity and yield from glucose in Escherichia coli devoid of the glucose phosphotransferase transport system

The effect of inactivation of the glucose phosphotransferase transport system (PTS) on 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) productivity and yield from glucose in Escherichia coli is reported. Strains used in this study were the PTS(+) PB103 and its PTS(-) glucose(+) derivative NF9. Their aroB(-) derivatives PB103B and NF9B were constructed to allow accurate measurement of total carbon flow into the aromatic pathway. The measured specific rates of DAHP synthesis were 0.55 and 0.94 mmol/g-dcw. h and the DAHP molar yields from glucose were 0.43 and 0.71 mol/mol for the PTS(+) aroB(-)and the PTS(-) glucose(+) aroB(-)strains, respectively. For the latter strain, this value represents 83% of the maximum theoretical yield for DAHP synthesis from glucose.     

Deletion mapping of the genes coding for HPr and enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system in Salmonella typhimurium

Sugars transported by a bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) require two soluble proteins: HPr, a low-molecular-weight phosphate-carrier protein, and enzyme I. The structural genes coding for HPr (ptsH) and Enzyme I (ptsI) are shown to be cotransducible in Salmonella typhimurium. The gene order of this region of the Salmonella chromosome is cysA-trzA-ptsH-ptsI...(crr). A method for the isolation of trzA-pts deletion is described. One class of pts deletions extends through ptsH and into ptsI; a second class includes both ptsH and ptsI and extends into or through the crr gene. The crr gene either codes for or regulates the synthesis of a third PTS protein (factor III) which is sugar-specific. A hypothesis is presented for a mechanism of deletion formation.     

Solubilization and reconstitution of the glucose transport system from Saccharomyces cerevisiae

The glucose transport system from Saccharomyces cerevisiae was solubilized from isolated plasma membranes by the nonionic detergent, octylglucoside. The transport system was reconstituted into proteoliposomes with removal of detergent from the extract by dialysis, followed by the addition of asolectin liposomes to the dialyzed proteins with a freeze-thaw and brief bath-sonication step. The reconstituted proteoliposomes exhibit specific carrier-mediated facilitated diffusion of d-glucose, including stimulated equilibrium exchange and influx counterflow. Furthermore, the reconstituted facilitated diffusion system shows substrate specificities similar to those of the intact cell d-glucose transport system.     

Reconstitution into liposomes of glucose active transport from the rabbit renal proximal tubule. Characteristics of the system

This paper describes the characteristics of Na⁺-dependent d-glucose transport into liposomes made from soybean phospholipids into which have been reconstituted detergent-solubilized components from the rabbit renal proximal tubular brush border membrane. Conditions for optimal and quantitative reconstitution of glucose carriers are defined. Na⁺-dependent d-glucose uptake occurs via a saturable system with a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 0.125–0.135 mM, is responsive to the volume of the internal liposomal space, and shows ‘overshoot’ as seen in natural membranes. The rate of Na⁺-dependent d-glucose uptake and the magnitude of the ‘overshoot’ are proportional to the concentration of protein used in reconstitution.     

肠杆菌共同抗原的研究进展

肠杆菌共同抗原(Enterobacterial common antigen,ECA)是由多糖重复单元组成的多聚糖,几乎表达于所有肠杆菌细菌外膜,具有生物学功能。ECA由多基因协同作用而合成,这些基因在肠杆菌细菌基因组上成簇存在,形成ECA抗原基因簇。ECA是重要的毒力因子,在肠杆菌细菌入侵宿主、体内存活等过程中有一定作用。同时,ECA在维持细菌外膜渗透屏障、鞭毛表达、群集运动及抗胆酸胆盐等方面也有重要作用。此外,锚定在细菌脂多糖核心区的ECALPS还是细菌重要的表面抗原,能激发宿主产生高水平抗体,可以作为疫苗研究的靶点。结合笔者的研究,文中对ECA纯化、基因结构和合成、免疫特性、生物学功能及应用等方面进行了综述。     

Effects of lipids on the transport activity of the reconstituted glucose transport system from rat adipocyte

The glucose transport system, isolated from rat adipocyte membrane fractions, was reconstituted into phospholipid vesicles. Vesicles composed of crude egg yolk phospholipids, containing primarily phosphatidylcholine (PC) and phosphatidylethanolamine (PE), demonstrated specific d-glucose uptake. Purified vesicles made of PC and PE also supported such activity but PC or PE by themselves did not. The modulation of this uptake activity has been studied by systematically altering the lipid composition of the reconstituted system with respect to: (1) polar headgroups; (2) acyl chains, and (3) charge. Addition of small amounts (20 mol%) of PS, phosphatidylinositol (PI), cholesterol, or sphingomyelin significantly reduced glucose transport activity. A similar effect was seen with the charged lipid, phosphatidic acid. In the case of PS, this effect was independent of the acyl chain composition. Polar headgroup modification of PE, however, did not appreciably affect transport activity. Free fatty acids, on the other hand, increased or decreased activity based on the degree of saturation and charge. These results indicate that glucose transport activity is sensitive to specific alterations in both the polar headgroup and acyl chain composition of the surrounding membrane lipids.     

A hydrolase-related transport system is not required to explain the intestinal uptake of glucose liberated from phlorizin

The fate of [³H]glucose released from a wide range of [³H]phlorizin concentrations by phlorizin hydrolase has been studied under conditions where the Na⁺-dependent glucose transport system in hamster intestine is profoundly inhibited by the glucoside. At 0.2–2.0 mM phlorizin, the [³H]glucose uptake was a constant 11–12% of that generated by the enzyme and at the highest level, it was reduced to that of passive diffusion. Glucose liberated from 0.2 mM [³H]phlorizin is accumulated at a rate nearly equal to that found for 0.2 mM [¹⁴C]glucose when this free sugar uptake is measured in a medium containing 0.2 mM unlabeled phlorizin. Furthermore, without sodium, the accumulation rates of hydrolase-derived or exogenous glucose are both reduced to the rate of [¹⁴C]mannitol. Our results indicate that the glucose released from phlorizin enters the tissue via the small fraction of the Na⁺-dependent glucose carriers which escape phlorizin blockade together with a mannitol-like passive diffusion. It enjoys a kinetic advantage for tissue entry over free glucose in the medium by virtue of the position of the site where it is formed, i.e. inside the unstirred water layer and near normal entry portals. No special hydrolase-related transport system, like the one proposed for disaccharides, needs to be considered to account for our findings.     

Non-PTS uptake and subsequent metabolism of glucose in Pediococcus halophilus as demonstrated with a double mutant defective in phosphoenolpyruvate:mannose phosphotransferase system and in phosphofructokinase

Pediococcus halophilus possesses phosphoenolpyruvate:mannose phosphotransferase system (man:PTS) as a main glucose transporter. A man:PTS defective (man:PTS^d) strain X-160 could, however, utilize glucose. A possible glucose-transport mechanism other than PTS was studied with the strain X-160 and its derivative, man:PTS^d phosphofructokinase defective (PFK^–) strain M-13. Glucose uptake by X-160 at pH 5.5 was inhibited by any of carbonylcyanide m-chlorophenylhydrazone, nigericin, N,N-dicyclohexylcarbodiimide, or iodoacetic acid. The double mutant M-13 could still transport glucose and accumulated intracellularly a large amount of hexose-phosphates (ca. 8 mM glucose 6-phosphate and ca. 2 mM fructose 6-phosphate). Protonophores also inhibited the glucose transport at pH 5.5, as determined by the amounts of accumulated hexose-phosphates (< 4 mM). These showed involvement of proton motive force (P) in the non-PTS glucose transport. It was concluded that the non-PTS glucose transporter operated in concert with hexokinase or glucokinase for the metabolism of glucose in the man:PTS^d strain.Abbreviations BM basal medium - BM-G basal medium containing glucose - CM complex medium - man:PTS phosphoenolpyruvate:mannose phosphotransferase system - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - P proton motive force - pH transmembrane pH gradient - transmembrane electrical potential difference - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PIPES piperazine-N,N-bis(-ethanesulfonic acid) - MES 4-morpholineethanesulfonic acid - G-6-P glucose 6-phosphate - F-6-P fructose 6-phosphate - FDP fructose 1,6-bisphosphate - EMP Embden-Meyerhof-Parnas pathway - PFK phosphofructokinase - GK glucokinase - HK hexokinase - IAA iodoacetic acid - II^man enzyme II component of man:PTS     

Characterization of glk, a gene coding for glucose kinase of Corynebacterium glutamicum

The glk gene from Corynebacterium glutamicum was isolated by complementation using Escherichia coli ZSC113 (ptsG ptsM glk). We sequenced a total of 3072 bp containing the 969-bp open reading frame encoding glucose kinase (Glk). The glk gene has a deduced molecular mass of 34.2 kDa and contains a typical ATP binding site. Comparison with protein sequences revealed homologies to Glk from Streptomyces coelicolor (43%) and Bacillus megaterium (35%). The glk gene in C. glutamicum was inactivated on the chromosome via single crossover homologous recombination and the resulting glk mutant was characterized. Interestingly, the C. glutamicum glk mutant showed poor growth on rich medium such as LB medium or brain heart infusion medium in the presence or absence of glucose, fructose, maltose or sucrose as the sole carbon source. Growth yield was reduced significantly when maltose was used as the sole carbon source using minimal medium. The growth defect of glk mutant on rich medium was complemented by a plasmid-encoded glk gene. A chromosomal glk-lacZ fusion was constructed and used to monitor glk expression, and it was found that glk was expressed constitutively under all tested conditions with different carbon sources.     

Evolutionary sequence divergence within repeated DNA families of higher plant genomes

The higher proportion of repeated DNA sequences in the garden pea (Pisum sativum) than in the mung bean (Vigna radiata), as well as other differences between these legume genomes, are consistent with a higher rate of sequence amplification in the former. This hypothesis leads to a prediction that repeated sequence families inPisum are mostly heterogeneous, as defined by Bendich and Anderson (1977), whileVigna families are homogeneous. An assay developed by these authors to distinguish between the two types of families, by comparison of reassociation rates at different temperatures, was utilized. The results forVigna defied the predictions of the assay for either homogeneous or hetereogeneous model. Evaluation of the kinetic data in light of the great diversity of repeated family copy numbers in both genomes enabled an interpretation of the results as consistent with hetereogenous families inPisum and homogeneous families inVigna. These tentative conclusions were supported by the results of a thermal denaturation (melting) assay described in the accompanying paper.Abbreviations used C_ot the product of molar concentration of DNA nucleotides and time of incubation (mol s/L) - EC_ot equivalent - C_ot the value after correction to standard reassociation conditions (120 mM sodium phosphate buffer, 60°C) - (Et)₄NCl tetraethylammonium chloride - T_m the temperature at which half of the nucleotides in solution are unpaired This paper is Carnegie Institution of Washington Department of Plant Biology Publication No. 708 and is based on a portion of a dissertation submitted by R.S.P. in partial fulfillment of the Ph.D. requirements at Stanford University     

Evolutionary analysis for functional divergence of Jak protein kinase domains and tissue-specific genes

Jak (Janus kinase) is a nonreceptor tyrosine kinase, which plays important roles in signal transduction pathways. The unique feature of Jak is that, in addition to a fully functional tyrosine kinase domain (JH1), Jak possesses a pseudokinase domain (JH2). Although JH2 lost its catalytic function, experimental evidence has shown that this domain may have acquired some new but unknown functions. This apparent functional divergence after the (internal) domain duplication may result in dramatic changes of selective constraints at some sites. We conducted a data analysis to test this hypothesis. Our result shows that shifted selective constraints (or shifted evolutionary rates) between the JH1 and the JH2 domains are statistically significant. Predicted amino acid sites by posterior analysis can be classified into two groups: very conserved in JH1 but highly variable in JH2, and vice versa. Moreover, we have studied the evolutionary pattern of four tissue-specific genes, Jak1, Jak2, Jak3, and Tyk2, which were generated in the early stages of vertebrates. We found that after the (first) gene duplication, site-specific rate shifts between Jak2/Jak3 and Jak1/Tyk are significant, presumably as a consequence of functional divergence among these genes. The implication of our study for functional genomics is discussed.     

Synthetic neomycin-kanamycin phosphotransferase, type II coding sequence for gene targeting in mammalian cells

The bacterial neomycin-kanamycin phosphotransferase, type II enzyme is encoded by the neo gene and confers resistance to aminoglycoside drugs such as neomycin and kanamycin-bacterial selection and G418-eukaryotic cell selection. Although widely used in gene targeting in mouse embryonic stem cells, the neo coding sequence contains numerous cryptic splice sites and has a high CpG content. At least the former can cause unwanted effects in cis at the targeted locus. We describe a synthetic sequence, sneo, which encodes the same protein as that encoded by neo. This synthetic sequence has no predicted splice sites in either strand, low CpG content, and increased mammalian codon usage. In mouse embryonic stem cells sneo expressability is similar to neo. The use of sneo in gene targeting experiments should substantially reduce the probability of unwanted effects in cis due to splicing, and perhaps CpG methylation, within the coding sequence of the selectable marker.