大肠杆菌PTS系统改造及重组菌生长性能测定

利用Red重组系统对野生大肠杆菌Escherichia coli磷酸烯醇式丙酮酸-糖磷酸转移酶系统(Phosphoenolpyruvate:carbohydrate phosphotransferase system,PTS)进行修饰改造,敲除PTS系统中关键组分EⅡCBGlc的编码基因(ptsG),磷酸组氨酸搬运蛋白HPr的编码基因(ptsI),同时敲入来源于运动发酵单胞菌Zymomonas mobilis的葡萄糖易化体(Glucose facilitator)编码基因(glf),构建重组大肠杆菌,比较测定并系统评价了基因敲除和敲入对细胞的生长、葡萄糖代谢和乙酸积累的影响。敲除基因ptsG和ptsI造成大肠杆菌PTS系统部分功能缺失,细胞生长受到一定限制,敲入glf基因后,重组大肠杆菌能够利用Glf-Glk(葡萄糖易化体-葡萄糖激酶)途径,消耗ATP将葡萄糖进行磷酸化并转运进入细胞。通过该途径转运葡萄糖能够提高葡萄糖利用效率,降低副产物乙酸生成,同时能够使更多的碳代谢流进入后续相关合成途径,预期能够提高相关产物产量。     

The complete phosphotransferase system in Escherichia coli

We here tabulate and describe all currently recognized proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and their homologues encoded within the genomes of sequenced E. coli strains. There are five recognized Enzyme I homologues and six recognized HPr homologues. A nitrogen-metabolic PTS phosphoryl transfer chain encoded within the rpoN and ptsP operons and a tri-domain regulatory PTS protein encoded within the dha (dihydroxyacetone catabolic) operon, probably serve regulatory roles exclusively. In addition to several additional putative regulatory proteins, there are 21 (and possibly 22) recognized Enzyme II complexes. Of the 21 Enzyme II complexes, 7 belong to the fructose (Fru) family, 7 belong to the glucose (Glc) family, and 7 belong to the other PTS permease families. All of these proteins are briefly described, and phylogenetic data for the major families are presented.     

9-Mercaptodethiobiotin is formed as a competent catalytic intermediate by Escherichia coli biotin synthase

Biotin synthase (BS) catalyzes the oxidative addition of a sulfur atom to dethiobiotin (DTB) to generate the biotin thiophane ring. This enzyme is an S-adenosylmethionine (AdoMet) radical enzyme that catalyzes the reductive cleavage of AdoMet, generating methionine and a transient 5'-deoxyadenosyl radical. In our working mechanism, the 5'-deoxyadenosyl radical oxidizes DTB by abstracting a hydrogen from C6 or C9, generating a dethiobiotinyl carbon radical that is quenched by a sulfide from a [2Fe-2S] (2+) cluster. A similar reaction sequence directed at the other position generates the second C-S bond in the thiophane ring. Since the BS active site holds only one AdoMet and one DTB, it follows that dissociation of methionine and 5'-deoxyadenosine and binding of a second equivalent of AdoMet must be intermediate steps in the formation of biotin. During these dissociation-association steps, a discrete DTB-derived intermediate must remain bound to the enzyme. In this work, we confirm that the conversion of DTB to biotin is accompanied by the reductive cleavage of 2 equiv of AdoMet. A discrepancy between DTB consumption and biotin formation suggests the presence of an intermediate, and we use liquid chromatography and mass spectrometry to demonstrate that this intermediate is indeed 9-mercaptodethiobiotin, generated at approximately 10% of the total enzyme concentration. The amount of intermediate observed is increased when the reaction is run with substoichiometric levels of AdoMet or with the defective enzyme containing the Asn153Ser mutation. The retention of 9-mercaptodethiobiotin as a tightly bound intermediate is consistent with a mechanism involving the stepwise radical-mediated oxidative abstraction of sulfide from an iron-sulfur cluster.     

The glucose-specific carrier of the Escherichia coli phosphotransferase system.

Thirteen glucose analogues bearing electrophilic groups were synthesized (five of them for the first time) and screened as inhibitors of the glucose transporter (EIIGlc) of the Escherichia coli phosphoenolpyruvate-sugar phosphotransferase system (PTS). 2',3'-Epoxypropyl beta-d-glucopyranoside (3a) is an inhibitor and also a pseudosubstrate. Five analogues are inhibitors of nonvectorial Glc phosphorylation by EIIGlc but not pseudosubstrates. They are selective for EIIGlc as demonstrated by comparison with EIIMan, another Glc-specific but structurally different transporter. 3a is the only analogue that inhibits EIIGlc by binding to the high-affinity cytoplasmic binding site and also strongly inhibits sugar uptake mediated by this transporter. The most potent inhibitor in vitro, methyl 6,7-anhydro-d,l-glycero-alpha-d-gluco-heptopyranoside (1d), preferentially interacts with the low-affinity cytoplasmic site but only weakly inhibits Glc uptake. Binding and/or phosphorylation from the cytoplasmic side of EIIGlc is more permissive than sugar binding and/or translocation of substrates via the periplasmic site. EIIGlc is rapidly inactivated by the 6-O-bromoacetyl esters of methyl alpha-d-glucopyranoside (1a) and methyl alpha-d-mannopyranoside (1c), methyl 6-deoxy-6-isothiocyanato-alpha-d-glucopyranoside (1e), beta-d-glucopyranosyl isothiocyanate (3c) and beta-d-glucopyranosyl phenyl isothiocyanate (3d). Phosphorylation of EIIGlc protects, indicating that inactivation occurs by alkylation of Cys421. Glc does not protect, but sensitizes EIIGlc for inactivation by 1e and 3d, which is interpreted as the effect of glucose-induced conformational changes in the dimeric transporter. Glc also sensitizes EIIGlc for inactivation by 1a and 1c of uptake by starved cells. This indicates that Cys421 which is located on the cytoplasmic domain of EIIGlc becomes transiently accessible to substrate analogues on the periplasmic side of the transporter.     

Identification of a phosphotransferase system of Escherichia coli required for growth on N-acetylmuramic acid

We report here that wild-type Escherichia coli grows on N-acetylmuramic acid (MurNAc) as the sole source of carbon and energy. Analysis of mutants defective in N-acetylglucosamine (GlcNAc) catabolism revealed that the catabolic pathway for MurNAc merges into the GlcNAc pathway on the level of GlcNAc 6-phosphate. Furthermore, analysis of mutants defective in components of the phosphotransferase system (PTS) revealed that a PTS is essential for growth on MurNAc. However, neither the glucose-, mannose/glucosamine-, nor GlcNAc-specific PTS (PtsG, ManXYZ, and NagE, respectively) was found to be necessary. Instead, we identified a gene at 55 min on the E. coli chromosome that is responsible for MurNAc uptake and growth. It encodes a single polypeptide consisting of the EIIB and C domains of a so-far-uncharacterized PTS that was named murP. MurP lacks an EIIA domain and was found to require the activity of the crr-encoded enzyme IIA-glucose (EIIA(Glc)), a component of the major glucose transport system for growth on MurNAc. murP deletion mutants were unable to grow on MurNAc as the sole source of carbon; however, growth was rescued by providing murP in trans expressed from an isopropylthiogalactopyranoside-inducible plasmid. A functional His(6) fusion of MurP was constructed, isolated from membranes, and identified as a polypeptide with an apparent molecular mass of 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Close homologs of MurP were identified in the genome of several bacteria, and we believe that these organisms might also be able to utilize MurNAc.     

Polyphosphate kinase from Escherichia coli. Purification and demonstration of a phosphoenzyme intermediate

Polyphosphate kinase (PPK) polymerizes the terminal phosphate of ATP to a long chain polyphosphate (poly(P) or (Pi)n) in a freely reversible reaction (Kornberg, S. R. (1957) Biochim. Biophys. Acta 26, 294-300), nATP in equilibrium nADP + (Pi)n, PPK, now purified to homogeneity, is a tetramer of 69-kDa subunits. Addition of a primer in the synthetic reaction is not required, nor does ATP or inorganic orthophosphate (Pi) serve in this role. PPK is autophosphorylated under the conditions of poly(P) synthesis; Pi is linked by a nitrogen-phosphate bond as judged by its acid lability and alkali stability. Incorporation of phosphate from the isolated phosphoenzyme into poly(P) upon the addition of ATP in the synthetic reaction and its incorporation into ATP upon the addition of ADP indicate phosphoenzyme to be an intermediate in the reaction. At an ATP level of 5 microM, well below its Km of 2 mM, a pronounced lag in poly(P) synthesis can be removed by tetrapolyphosphate but not by Pi, PPi, or tripolyphosphate. The basis for this stimulatory effect is not clear inasmuch as tetrapolyphosphate does not promote the dephosphorylation of the presumed phosphoenzyme intermediate.     

An inducible phosphoenolpyruvate: dihydroxyacetone phosphotransferase system in Escherichia coli

A phosphoenolpyruvate: dihydroxyacetone phosphotransferase was induced in Escherichia coli grown on dihydroxyacetone as sole carbon source or in its presence. This is the first example of a triose which can be acted upon by the membrane complex to provide a central intermediate in glycolysis. The presence of this system explains the ability of a mutant, in which the ATP-dependent glycerol kinase is genetically replaced by a glycerol: NAD 2-oxidoreductase, to grow on glycerol.     

Mannitol-specific carrier protein from the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system can be extracted as a dimer from the membrane

The association state of the mannitol-specific enzyme II (EIIMtl) has been studied both in the purified form and embedded in the cytoplasmic membrane. Membrane fragments obtained from mannitol-grown Escherichia coli catalyze the phosphoenolpyruvate- (PEP) dependent phosphorylation of both glucose and mannitol; thus they contain both the glucose- and mannitol-specific enzymes II. The autoradiogram of an electrophoresed mixture of [32P]PEP, EI, HPr, and membrane fragments shows bands at 58 and 116 kilodaltons, in addition to the bands of P-EI and P-HPr. In an analogous experiment with purified EIIMtl, suspended in detergent micelles, only a 58 000-dalton band and the P-HPr and P-EI bands were found. Treatment of the phosphorylated membranes with mannitol results in an immediate substantial decrease in the radioactivity in the 58- and 116-kilodalton bands. A similar treatment of the phosphorylated membranes with glucose had no direct effect on the autoradiogram. We conclude therefore that the 58- and 116-kilodalton bands originate from enzyme IIMtl monomers and dimers, respectively. The interaction between the subunits of the dimer is not abolished by the addition of up to 5% sodium dodecyl sulfate. However, the nonionic detergent Lubrol PX, which is present during the purification of EIIMtl, is capable of transforming the enzyme IIMtl dimers into monomers.     

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.     

Engineering transport protein function: theoretical and technical considerations using the sugar-transporting phosphotransferase system of Escherichia coli as a model system

Potential experimental approaches for developing and applying protein-engineering protocols to transmembrane transport systems are described. We specifically consider procedures designed to alter protein function. These procedures are designed for the specific purposes of (1) changing protein interaction specificities and (2) changing a protein's catalytic function. We use sugar-transporting bacterial phosphotransferase systems as model systems to illustrate the proposed approaches. These and other similar procedures are likely to prove to be of utility for biotechnological manipulation of proteins as well as for elucidating potential evolutionary pathways taken for the appearance of novel functions within a protein family.     

Lactose transport mutants of Escherichia coli resistant to inhibition by the phosphotransferase system

The lactose carrier activity of Escherichia coli is inhibited by the binding of dephosphorylated glucose enzyme III. Saier et al. ((1978) J. Bacteriol. 133, 1358-1367) isolated lacY mutants that escaped this inhibition. This communication reports the cloning and sequencing of one of the Saier mutants and the isolation, cloning and sequencing of another similar mutant. Both mutations resulted in amino acid substitutions on the middle cytoplasmic loop of the carrier (alanine-198 to valine and serine-209 to isoleucine). It is concluded that this cytoplasmic loop may be one of the sites of binding of glucose enzyme III.