Publications

• The mannose transporter of Escherichia coli K12: oligomeric structure, and function of two conserved cysteines.

  Rhiel E., Flukiger K., Wehrli C., Erni B.

  The mannose transporter of E. coli is a member of the phosphotransferase system. It consists of two membrane spanning subunits, IICMan (27.64 kDa) and IIDMan (31.02 kDa) and a peripheral subunit IIABMan (35.02 kDa). It acts by a mechanism that couples vectorial translocation to phosphorylation of ... >> More

  The mannose transporter of E. coli is a member of the phosphotransferase system. It consists of two membrane spanning subunits, IICMan (27.64 kDa) and IIDMan (31.02 kDa) and a peripheral subunit IIABMan (35.02 kDa). It acts by a mechanism that couples vectorial translocation to phosphorylation of the substrate. The subunit ratio determined from densitometric scans of polyacrylamide gels is close to IIABMan2 IICMan1 IIDMan2. A molecular mass of 100 +/-20 kDa was calculated from electronmicrographs of freeze fractured proteoliposomes containing particles of the IICMan/IIDMan subcomplex with a mean diameter of 6.3 +/-1.1 nm. This is most compatible with IICMan:IIDMan subunit compositions of 1:2 (89.7 kDa). Fusion proteins between IICMan and IIDMan were generated, with the subunits connected either by a two-residue linker or a 20 residue Ala Pro rich hinge. The fusion proteins had 5%-15% of control phosphotransferase activity. The one with the Ala Pro rich linker could be cleaved with trypsin resulting in a 7 fold increase of activity while the fusion with the two residue linker was resistant to limited trypsinolysis. Taking into account the inside-out orientation of the membrane vesicles the C-terminus of IICMan and the N-terminus of IIDMan are both predicted to be on the cytoplasmic side of the membrane. Two cysteines in IICMan and IIDMan which are conserved in the homologous subunits of the fructose transporter of Bacillus subtilis and of sorbose transporter of Klebsiella pneumoniae are not necessary for phosphotransferase function. << Less

  Biol. Chem. Hoppe-Seyler 375:551-559(1994) [PubMed] [EuropePMC]

• Pel, the protein that permits lambda DNA penetration of Escherichia coli, is encoded by a gene in ptsM and is required for mannose utilization by the phosphotransferase system.

  Williams N., Fox D.K., Shea C., Roseman S.

  Mannose uptake and phosphorylation in Escherichia coli is catalyzed by the phosphoenolpyruvate:glycose phosphotransferase system (PTS). The mannose-specific complex of the PTS, designated IIMan, comprises lipid and two membrane proteins, II-AMan and II-BMan. The proteins are encoded by ptsM, locat ... >> More

  Mannose uptake and phosphorylation in Escherichia coli is catalyzed by the phosphoenolpyruvate:glycose phosphotransferase system (PTS). The mannose-specific complex of the PTS, designated IIMan, comprises lipid and two membrane proteins, II-AMan and II-BMan. The proteins are encoded by ptsM, located at approximately equal to 40 minutes on the E. coli chromosome. A different genetic marker, pel, maps with ptsM, and is required for lambda DNA penetration of the cytoplasmic membrane. Earlier studies suggested that both pel function and II-BMan are encoded by the same gene, while a different gene (also in ptsM) encodes II-AMan. In the present studies, a ptsM clone, pCS13, was isolated from an E. coli HindIII gene bank in pBR322 and restored both mannose termentation and pel+ function to ptsM mutants defective in II-BMan. Subclones of pCS13 show that two distinct genes, manY and manZ, encode the pel+ function and the II-BMan protein, respectively; each gene may have its own promoter; whereas the protein encoded by manY (Pel) alone seems sufficient for lambda sensitivity, all three gene products are required for mannose fermentation, transport of the mannose analogue 2-deoxyglucose, and phosphorylation of the latter by cytoplasmic membranes. Thus, Pel is required for function of the IIMan complex. The efficiency of the complex may depend on the ratio of Pel to IIMan. << Less

  Proc Natl Acad Sci U S A 83:8934-8938(1986) [PubMed] [EuropePMC]

• The mannose transporter of Escherichia coli. Structure and function of the IIABMan subunit.

  Stolz B., Huber M., Markovic-Housley Z., Erni B.

  <h4>Unlabelled</h4>The mannose transporter of the bacterial phosphotransferase system consists of two transmembrane subunits (IICMan and IIDMan) and a hydrophilic subunit (IIABMan). IIABMan has two flexibly linked domains containing one phosphorylation site each and occurs as a dimer. Substrate tr ... >> More

  <h4>Unlabelled</h4>The mannose transporter of the bacterial phosphotransferase system consists of two transmembrane subunits (IICMan and IIDMan) and a hydrophilic subunit (IIABMan). IIABMan has two flexibly linked domains containing one phosphorylation site each and occurs as a dimer. Substrate transport is coupled to phosphorylation. The phosphoryl group is transferred from a phosphoryl carrier protein to His10 on IIA, hence to His175 on IIB and finally to the substrate. IIABMan mutants were analyzed in vitro for complementation, negative dominance, cysteine cross-linking and reactivity.<h4>Conclusions</h4>(i) His10, Trp12, Lys48, and Ser72 form a functional unit (phosphorylation site 1); (ii) His86 on the IIA domain and His175 on the IIB domain of the same subunit form a functional unit (phosphorylation site 2); (iii) phosphoryl transfer can occur between His10 and His175 of the same as well as of different subunits and His86 is necessary for this transfer; (iv) the subunits in the dimer are interdependent; (v) The phosphorylation site mutant H175C is highly reactive toward thiol reagents and it forms extensive homo- and heterocross-links with other surface-exposed cysteines. The phosphorylation site mutant H10C is 1000-fold less reactive. The two residues might be in complementary locations, His10 buried in a concave, His175 exposed on a convex surface. << Less

  J. Biol. Chem. 268:27094-27099(1993) [PubMed] [EuropePMC]

• The mannose-permease of the bacterial phosphotransferase system. Gene cloning and purification of the enzyme IIMan/IIIMan complex of Escherichia coli.

  Erni B., Zanolari B.

  The mannose-permease complex of the phosphoenolpyruvate-dependent phosphotranferase system exhibits two apparently unrelated activities. It mediates active transport concomitant with phosphorylation of mannose, 2-deoxyglucose, and a number of other hexoses, and it is required for penetration of ba ... >> More

  The mannose-permease complex of the phosphoenolpyruvate-dependent phosphotranferase system exhibits two apparently unrelated activities. It mediates active transport concomitant with phosphorylation of mannose, 2-deoxyglucose, and a number of other hexoses, and it is required for penetration of bacteriophage lambda DNA across the cytoplasmic membrane of Escherichia coli. A cloned fragment of E. coli chromosome restores mannose-fermentation in, and confers lambda sensitivity to, an E. coli strain with a mutation in the gene for the phosphoenolpyruvate-dependent mannose uptake. Using complementation analysis of phosphotransferase activity and of lambda sensitivity, a 3.8-kilobase pair fragment was shown to carry two adjacent genes, ptsM and ptsL. Although each gene has a promoter of its own, transcription of ptsL has a positive polar effect on the transcription of ptsM. A complex of two proteins, IIMan and IIIMan, was purified to homogeneity from an overproducing strain. IIMan is encoded by gene ptsM, IIIMan by gene ptsL. IIMan, a 27-kDa protein, is the transmembrane component of the complex. IIIMan, a 35-kDa protein, exists as a dimer and is found both membrane-associated and free in the cytoplasm. IIIMan can be phosphorylated in a phosphoenolpyruvate-dependent reaction, while phosphorylation of IIMan could not be detected. IIMan and IIIMan are both required for phosphorylation of 2-deoxyglucose in vitro, while IIMan alone is sufficient to confer lambda sensitivity. << Less

  J. Biol. Chem. 260:15495-15503(1985) [PubMed] [EuropePMC]

• The mannose permease of Escherichia coli consists of three different proteins. Amino acid sequence and function in sugar transport, sugar phosphorylation, and penetration of phage lambda DNA.

  Erni B., Zanolari B., Kocher H.P.

  The mannose permease of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It also functions as a receptor for bacterial chemotaxis and is required for infection of the cell by bacteriophage lambda where it most ... >> More

  The mannose permease of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It also functions as a receptor for bacterial chemotaxis and is required for infection of the cell by bacteriophage lambda where it most likely functions as a pore for penetration of lambda DNA. The permease consists of three different subunits, IIIMan, II-PMan, and II-MMan, which are encoded in a single transcriptional unit ptsLPM. The complete amino acid sequence of the subunits is deduced from the nucleotide sequence. IIIMan (35 kDa) is a hydrophilic protein which is transiently phosphorylated and most likely contains the active site for sugar phosphorylation. II-PMan (28 kDa) is very hydrophobic; II-MMan (31 kDa) is moderately hydrophobic. Both are integral membrane proteins and most likely form the transmembrane channel. All three subunits are required for sugar transport and phosphorylation; II-PMan and II-MMan alone are sufficient for penetration of lambda DNA. Truncated forms of II-MMan and II-PMan are described that mediate lambda DNA penetration but have no apparent sugar transport activity. Residual sugar phosphorylation activity is found with the truncated form of II-PMan. No obvious homologies at the level of amino acid sequence could be detected with other bacterial transport proteins. << Less

  J. Biol. Chem. 262:5238-5247(1987) [PubMed] [EuropePMC]

• Membrane topology of the mannose transporter of Escherichia coli K12.

  Huber F., Erni B.

  The mannose transporter of the bacterial phosphotransferase system mediates carbohydrate transport across the cytoplasmic membrane concomitant with carbohydrate phosphorylation. It also functions as a receptor for bacterial chemotaxis [Adler.J. & Epstein, W. (1974) Proc. Natl Acad. Sci. USA 71. 28 ... >> More

  The mannose transporter of the bacterial phosphotransferase system mediates carbohydrate transport across the cytoplasmic membrane concomitant with carbohydrate phosphorylation. It also functions as a receptor for bacterial chemotaxis [Adler.J. & Epstein, W. (1974) Proc. Natl Acad. Sci. USA 71. 2895-2899] and is required for infection of the cell by bacteriophage lambda where it most likely functions as a pore for penetration of phage DNA [Elliott, J. & Arber, W. (1978) Mol. & Gen. Genet. 161, 1-8]. The transporter consists of two transmembrane subunits (27-kDa IICMan and 31-kDa IIDMan) and a hydrophilic subunit (35-kDa IIABMan). Protein fusions of IICMan and IIDMan with beta-galactosidase (LacZ) and with alkaline phosphatase (PhoA) were analyzed to determine the membrane topology of the two proteins. Protein fusions were obtained by progressively deleting the manY and manZ genes from their 3' ends and ligating them to lacZ and 'phoA that lack promotor and leader sequences. Based on the analysis of 30 IICMan-PhoA. 10 IICMan-LacZ, 12 IIDMan-PhoA, and 30 IIDMan-LacZ fusions, it is predicted that IICMan has six membrane-spanning segments with the N- and C-termini on the cytoplasmic face of the membrane. IIDMan is anchored in the membrane by a single membrane-spanning segment at the end of the C-terminus, while most of the protein (250 residues) protrudes into the cytoplasm. << Less

  Eur. J. Biochem. 239:810-817(1996) [PubMed] [EuropePMC]