Abstract
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.
Highlights
Both glucose permease and manphorylation.It alsofunctionsas a receptor for bacterianl ose permease are componentsof the bacterial phosphotransferase system
The five recombinant plasmids (Fig. 4a) were used in complementation experiments with two E. coli strains, WA2127 which is deficient in all three subunits of the mannose permease and WA3155 which expresses IIIMa"and II-MMa"but lacks functional 11-PMa"T. ransformants were analyzed (i) for fermentation of mannose on indicator plates, a qualitative assay to detect sugar transport by intact cells (Fig. 5a); (ii) for phosphotransferase activity of membrane preparations, a quantitative assay for permease activity (Figs. 5b and 6); and (iii) for infection by phage X (Fig. 5,a and c)
On 2127(pTSPM26) the efficiency of plating is reduced 100-fold (Law2, open bar) and the plaque diameter is reduced (0.6 mm instead of1.5-1.7 mm, not shown). d, titration of Xvir hp 8 on WA2127 and WA3155 (Ames, 1986); sugar:ion cotransport (Wrightet al., 1986; Kaback, 1986); and group translocation (Postma and Lengeler, 1985;Saier, 1985).So far all shock-sensitivetransport systems were found to consist of a substrate-binding protein located in the periplasm andthree cytoplasmic membrane-bound transformed with plasmids indicated at the top
Summary
AMINOACID SEQUENCE AND FUNCTIONIN SUGAR TRANSPORT, SUGAR PHOSPHORYLATION, AND PENETRATION OF PHAGE X DNA*. Because this latterpermease is required for rapid transferase system mediates sugar transport acrossgrtohweth on mannose, it is termed mannose permease (gene cytoplasmic membrane concomitant with sugar phos- product of the ptsM locus). The first open reading frame (gene ptsL) in which a GTG codon initiates translation encodes IIIMa",the cytoplasmic component of the mannose permease complex. The dimeric form of,"'II orientation and stoichiometry of pairs downstream by an ATG codonthat initiates translation of ptsP This gene encodes 11-PMa"a, very hydrophobic 28kDa protein. II-PMa" and II-M'"" neither of whichcouldbe detected in proteins in a single step by phosphocellulose chromatography phosphorylated form (Erni andZanolari, 1985) are hydropho- (Fig. 9, lane 10).A complex of II-PM""and II-""" was purified bic and together most likely constitute the transmembrane by isoelectric focusing and gel filtration, the same methods channel of the mannose permease (Fig. 1).
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