A single mutation in enzyme I of the sugar phosphotransferase system confers penicillin tolerance to Streptococcus gordonii.

Tolerance is a poorly understood phenomenon that allows bacteria exposed to a bactericidal antibiotic to stop their growth and withstand drug-induced killing. This survival ability has been implicated in antibiotic treatment failures. Here, we describe a single nucleotide mutation (tol1) in a tolerant Streptococcus gordonii strain (Tol1) that is sufficient to provide tolerance in vitro and in vivo. It induces a proline-to-arginine substitution (P483R) in the homodimerization interface of enzyme I of the sugar phosphotransferase system, resulting in diminished sugar uptake. In vitro, the susceptible wild-type (WT) and Tol1 cultures lost 4.5 and 0.6 log(10) CFU/ml, respectively, after 24 h of penicillin exposure. The introduction of tol1 into the WT (WT P483R) conferred tolerance (a loss of 0.7 log(10) CFU/ml/24 h), whereas restitution of the parent sequence in Tol1 (Tol1 R483P) restored antibiotic susceptibility. Moreover, penicillin treatment of rats in an experimental model of endocarditis showed a complete inversion in the outcome, with a failure of therapy in rats infected with WT P483R and the complete disappearance of bacteria in animals infected with Tol1 R483P.

Initiation of mammalian protein synthesis: dynamic properties of the assembly process in vitro.

Binding of the Met-tRNAMetf . eIf-2 GTP complex to the 40 S ribosomal subunit is the first step in initiation of eukaryotic protein synthesis. The extent of binding and the stability of the complex are enhanced by initiation factors eIF-3 and eIF-4C, AUG and elevated magnesium concentration. The reversibility of reaction steps occurring during the assembly of the initiation complex is measured as the rate of Met-tRNAMetf exchange in the initiation complex and its intermediates. This rate progressively decreases and Met-tRNAMetf binding becomes irreversible upon binding of mRNA. The association of the 40 S Met-tRNAMetf mRNA initiation complex with the 60 S ribosomal subunit is again reversible as long as elongation does not occur.

Purification of seven protein synthesis initiation factors from Krebs II ascites cells.

Seven protein synthesis initiation factors were isolated from Krebs II ascites cells using the procedures developed for the purification of the corresponding factors from rabbit reticulocytes. The ascites factors display identical characteristics in ion exchange chromatography and sucrose density gradient sedimentation. Based on their profiles in SDS polyacrylamide gels, the ascites factors have polypeptide profiles and molecular weights identical to those of the reticulocyte factors. Most significantly, each ascites factor is competent in replacing its corresponding reticulocyte factor in a reconstituted in vitro protein synthesizing system which is dependent on all seven factors.

Crystal structure of the IIB subunit of a fructose permease (IIBLev) from Bacillus subtilis.

The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates both the uptake of carbohydrates across the cytoplasmic membrane and their phosphorylation. During this process, a phosphoryl group is transferred from phosphoenolpyruvate via the general PTS proteins enzyme I, HPr and the sugar-specific components IIA, IIB to the transported sugar. The crystal structure of the IIB subunit of a fructose transporter from Bacillus subtilis (IIBLev) was solved by MIRAS to a resolution of 2.9 A. IIBLev comprises 163 amino acid residues that are folded into an open, mainly parallel beta-sheet with helices packed on either face. The phosphorylation site (His15) is located on the first loop (1/A) at one of the topological switch-points of the fold. Despite different global folds, IIBLev and HPr have very similar active-site loop conformations with the active-site histidine residues located close to the N terminus of the first helix. This resemblance may be of functional importance, since both proteins exchange a phosphoryl group with the same IIA subunit. The structural basis of phosphoryl transfer from HPr to IIAMan to IIBMan was investigated by modeling of the respective transition state complexes using the known HPr and IIAMan structures and a homology model of IIBMan that was derived from the IIBLev structure. All three proteins contain a helix that appears to be suitable for stabilization of the phospho-histidine by dipole and H-bonding interactions. Smooth phosphoryl transfer from one N-cap position to the other appears feasible with a minimized transition state energy due to simultaneous interactions with the donor and the acceptor helix.

Crystallization and preliminary X-ray diffraction studies of the N-terminal domain of the phosphorylating subunit of mannose permease from Escherichia coli.

Mannose permease is a constitutive component of the phosphotransferase system in Escherichia coli. This complex consists of two transmembrane subunits (II-PMan, Mr = 28,000 and II-MMan, Mr = 31,000) and a hydrophilic subunit (IIIMan). IIIMan functions as a phosphorylating enzyme and exists as a soluble homo-dimer of Mr = 70,000 in the cytosol. The N-terminal domain (P13) of IIIMan contains a phosphorylation site and the interface for dimerization. P13 has been crystallized in two different forms: type I, orthorhombic, space group C222 with a = 98.7 A, b = 106.5 A and c = 57.4 A, and type II, monoclinic, space group P2(1), with a = 54.4 A, b = 100.5 A, c = 58.1 A and beta = 90.5 degrees. Both types of crystal are suitable for X-ray diffraction studies.

Structure of the IIA domain of the mannose transporter from Escherichia coli at 1.7 angstroms resolution.

The mannose transporter from Escherichia coli is a member of the phosphoenolpyruvate-dependent phosphotransferase system. The multi-subunit complex couples translocation across the bacterial inner membrane with phosphorylation of the solute. A functional fragment (IIA(Man), residues 2 to 133) of the membrane-associated IIAB(Man) subunit of the mannose transporter was expressed as a selenomethionine protein, and the unphosphorylated molecule was crystallized and its structure solved by X-ray crystallography. The protein consists of a central five-stranded beta-sheet covered by helices on either face. The order of the secondary structure elements is (beta alpha)4, alpha beta. Four beta-strands are arranged in a parallel manner with strand order 2134 and are linked by helices forming right-handed cross-over connections. The fifth strand that forms one edge of the sheet and runs antiparallel to the others is swapped between the subunits of the dimeric structure. Helices D and E form a helical hairpin. Histidine 10, which is transiently phosphorylated during catalysis, is located at the topological switch-point of the structure, close to the subunit interface. Its imidazole ring is hydrogen bonded to the buried side-chain of Asp67. It is likely that Asp67 acts as a general base and thus increases the nucleophilicity of the histidine. Modeling suggests that the covalently bound phosphoryl group would be stabilized by the macrodipole of helix C. Putative interactions between IIA(Man) and the histidine-containing phosphocarrier protein are discussed.

A functional protein hybrid between the glucose transporter and the N-acetylglucosamine transporter of Escherichia coli.

The glucose and N-acetylglucosamine-specific transporters (IIGlc/IIIGlc and IIGlcNAc) of the bacterial phosphotransferase system mediate carbohydrate uptake across the cytoplasmic membrane concomitant with substrate phosphorylation. The two transporters have 40% amino acid sequence identity. Eight chimeric proteins between the two transporters were made by gene reconstruction. All hybrid proteins could be expressed, some inhibited cell growth, and one was active. The active hybrid transporter consists of the transmembrane domain (residues 1-386) of the IIGlc subunit and the two hydrophilic domains (residues 370-648) of IIGlcNAc. The N-terminal hydrophilic domain of IIGlcNAc contains the transiently phosphorylated cysteine-412. The hybrid protein is specific for glucose, which indicates that the sugar specificity determinant is in the transmembrane domain and that the cysteine from which the phosphoryl group is transferred to the substrate is not part of the binding site. The protein sequence (LKTPGRED) at which the successful fusion occurred has the characteristic properties of an interdomain oligopeptide linker (Argos, P., 1990, J. Mol. Biol. 211, 943-958).

Effects of tryptophan to phenylalanine substitutions on the structure, stability, and enzyme activity of the IIAB(Man) subunit of the mannose transporter of Escherichia coli.

The hydrophilic subunit of the mannose transporter (IIAB(Man)) of Escherichia coli is a homodimer that contains four tryptophans per monomer, three in the N-terminal domain (Trp12, Trp33, and Trp69) and one in the C-terminal domain (Trp182). Single and double Trp-Phe mutants of IIABMan and of the IIA domain were produced. Fluorescence emission studies revealed that Trp33 and Trp12 are the major fluorescence emitters, Trp69 is strongly quenched in the native protein and Trp182 strongly blue shifted, indicative of a hydrophobic environment. Stabilities of the Trp mutants of dimeric IIA(Man) and IIAB(Man) were estimated from midpoints of the GdmHCl-induced unfolding transitions and from the amount of dimers that resisted dissociation by SDS (sodium dodecyl sulfate), respectively. W12F exhibited increased stability, but only 6% of the wild-type phosphotransferase activity, whereas W33F was marginally and W69F significantly destabilized, but fully active. Second site mutations W33F and W69F in the background of the W12F mutation reduced protein stability and suppressed the functional defect of W12F. These results suggest that flexibility is required for the adjustments of protein-protein contacts necessary for the phosphoryltransfer between the phosphorylcarrier protein HPr, IIA(Man), IIB(Man), and the incoming mannose bound to the transmembrane IIC(Man)-IID(Man) complex.

Base-pair formation between 18 S ribosomal RNA and globin mRNA during initiation of protein synthesis in vitro.

A stable complex between 18 S rRNA and globin mRNA has been isolated from 40 S initiation complexes in the reconstituted reticulocyte cell free system. This complex is only formed under the conditions which also lead to an initiation complex active in protein synthesis. The mRNA-18 S rRNA interaction has properties compatible with base-pairing. This observation is discussed in the context with other, in part controversial, observations relating to base pairing as a step in initiation of eukaryotic protein synthesis.

The glucose transporter of Escherichia coli. Purification and characterization by Ni+ chelate affinity chromatography of the IIBCGlc subunit.

The IIBCGlc transmembrane subunit of the glucose transporter of E. coli containing a carboxy-terminal affinity tag consisting of six adjacent histidines was purified by nickel chelate affinity chromatography. The protein was constitutively overexpressed from a high copy number plasmid. 1.5 mg of 95% pure protein was obtained from 5 g (wet weight) cells. 70% of the phosphotransferase activity present in cell membranes was recovered. Adsorption to the nickel resin allows delipidation as well as rapid detergent exchange. The procedure was used to demonstrate exchange of subunits in the IIBCGlc dimer and it holds promise for the investigation of other protein-protein interactions.

Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS).

The glucose transporter of Escherichia coli couples translocation with phosphorylation of glucose. The IICB(Glc) subunit spans the membrane eight times. Split, circularly permuted and cyclized forms of IICB(Glc) are described. The split variant was 30 times more active when the two proteins were encoded by a dicistronic mRNA than by two genes. The stability and activity of circularly permuted forms was improved when they were expressed as fusion proteins with alkaline phosphatase. Cyclized IICB(Glc) and IIA(Glc) were produced in vivo by RecA intein-mediated trans-splicing. Purified, cyclized IIA(Glc) and IICB(Glc) had 100% and 30% of wild-type glucose phosphotransferase activity, respectively. Cyclized IIA(Glc) displayed increased stability against temperature and GuHCl-induced unfolding.

Secondary structure of the IIB domain of the Escherichia coli mannose transporter, a new fold in the class of alpha/beta twisted open-sheet structures.

The mannose transporter of the Escherichia coli bacterial phosphotransferase system consists of three subunits: IIAB, IIC and IID. IIABMan transfers phosphoryl groups to the transported substrate via phosphohistidine intermediates. Its IIB domain was overexpressed and isotopically labelled with 13C, 15N and 2H. Heteronuclear 3D triple-resonance NMR experiments combined with a semi-automatic assignment procedure yielded the sequential assignment of the 1H, 13C and 15N backbone resonances. Based on the evaluation of conformationally sensitive parameters, the secondary structure of the IIBMan domain has been determined as an alpha/beta twisted open-sheet structure consisting of a six-stranded parallel beta-sheet with the novel strand order 3-2-4-1-5-6, six helices and a short two-stranded antiparallel beta-sheet.

Bacterial proteins with N-terminal leader sequences resembling mitochondrial targeting sequences of eukaryotes.

Amphipathic, alpha-helical, leader sequences, analogous to those that direct nuclear-encoded eukaryotic proteins into mitochondria, have been found in one and only one class of bacterial integral membrane proteins. These bacterial proteins are the sugar permeases of the phosphoenolpyruvate-dependent phosphotransferase system. The amphipathic leader sequence in each of these proteins is terminated by a helix breaker, either a prolyl residue or 2 adjacent glycyl residues. Preliminary evidence suggests that these leader sequences function to target the proteins to the envelope fraction of the prokaryotic cell during their biosynthesis.

Phospholipids containing photoactivable groups in studies of biological membranes.

Photoactivable carbene precursors, aryl diazirines and trifluorodiazopropionates, were incorporated synthetically into the omega-positions of fatty acids, which were used to synthesize phospholipids. Extensive intermolecular C--H insertion reactions were demonstrated by photolysis of liposomes prepared from the above phospholipids. Structural analysis of the cross-linked products showed that the predominant sites of cross-linking were in the expected positions within the bilayer. Studies on the topography of a number of membrane proteins using the above phospholipids were initiated. Cross-linking of the photoactivable phospholipids to membrane-embedded proteins, glycophorin A, cytochrome b5, and gramicidin A, was demonstrated.