Cysteine substitutions for individual residues in helix VI of the melibiose carrier of Escherichia coli.

The melibiose carrier of Escherichia coli is a cytoplasmic membrane protein that mediates the cotransport of galactosides with H(+), Na(+), or Li(+). In this study we used cysteine-scanning mutagenesis to try to gain information about the position of transmembrane helix VI in the three-dimensional structure of the melibiose carrier. We constructed 23 individual cysteine substitutions in helix VI and an adjacent loop of the carrier. The resulting melibiose carriers retained 22-100% of their ability to transport melibiose. We tested the effect of the hydrophilic sulfhydryl reagent p-chloromercuri-benzenesulfonic acid (PCMBS) on the cysteine-substitution mutants and we found that there was no inhibition of melibiose transport in any of the mutants. We suggest that helix VI is imbedded in phospholipid and does not face the aqueous channel through which melibiose passes.

Melibiose carrier of Escherichia coli: use of cysteine mutagenesis to identify the amino acids on the hydrophilic face of transmembrane helix 2.

The melibiose carrier from Escherichia coli is a galactoside-cation symporter. Based on both experimental evidence and hydropathy analysis, 12 transmembrane helices have been assigned to this integral membrane protein. Transmembrane helix 2 contains several charged and polar amino acids that have been shown to be essential for the cation-coupled transport of melibiose. Starting with the cysteine-less melibiose carrier, we have individually substituted cysteine for amino acids 39-66, which includes the proposed transmembrane helix 2. In the resulting derivative carriers, we measured the transport of melibiose, determined the effect of the hydrophilic sulfhydryl reagent, p-chloromercuribenzenesulfonic acid (PCMBS), on transport in intact cells and inside out vesicles, and examined the ability of melibiose to protect the carrier from inactivation by the sulfhydryl reagent. We found a set of seven positions in which the reaction with the sulfhydryl reagent caused partial or complete loss of carrier function measured in intact cells or inside-out vesicles. The presence of melibiose protected five of these positions from reaction with PCMBS. The reaction of two additional positions with PCMBS resulted in the partial loss of transport function only in inside-out vesicles. Melibiose protected these two positions from reaction with the reagent. Together, the PCMBS-sensitive sites and charged residues assigned to helix 2 form a cluster of amino acids that map in three rows with each row comprised of every fourth residue. This is the pattern expected of residues that are part of an alpha-helical structure and thus the rows are tilted at an angle of 25 degrees to the helical axis. We suggest that these residues line the path of melibiose and its associated cation through the carrier.

The construction of a cysteine-less melibiose carrier from E. coli.

The melibiose carrier of E. coli is a cation-sugar cotransport system. This membrane protein contains four cysteine residues and the transport function is inhibited by sulfhydryl reagents. In order to investigate the importance of the cysteines, we have constructed a set of four melibiose transporters each of which has one cysteine replaced with serine or valine. The sensitivity of this set of carriers to N-ethylmaleimide was tested and Cys364 was identified as the target of the reagent. In addition, we constructed a melibiose transporter in which all 4 cysteines were replaced with either serine (Cys110, Cys310, and Cys364) or valine (Cys235) and we found that, as expected, the resulting cysteine-less transporter was resistant to the action of N-ethylmaleimide. The cysteine-less melibiose carrier had no significant decrease in ability to accumulate melibiose with cotransported sodium ions or protons. Thus, none of the 4 cysteines are necessary for the function of the melibiose carrier.

Domains of Escherichia coli acyl carrier protein important for membrane-derived-oligosaccharide biosynthesis.

Acyl carrier protein participates in a number of biosynthetic pathways in Escherichia coli: fatty acid biosynthesis, phospholipid biosynthesis, lipopolysaccharide biosynthesis, activation of prohemolysin, and membrane-derived oligosaccharide biosynthesis. The first four pathways require the protein's prosthetic group, phosphopantetheine, to assemble an acyl chain or to transfer an acyl group from the thioester linkage to a specific substrate. By contrast, the phosphopantetheine prosthetic group is not required for membrane-derived oligosaccharide biosynthesis, and the function of acyl carrier protein in this biosynthetic scheme is currently unknown. We have combined biochemical and molecular biological approaches to investigate domains of acyl carrier protein that are important for membrane-derived oligosaccharide biosynthesis. Proteolytic removal of the first 6 amino acids from acyl carrier protein or chemical synthesis of a partial peptide encompassing residues 26 to 50 resulted in losses of secondary and tertiary structure and consequent loss of activity in the membrane glucosyltransferase reaction of membrane-derived oligosaccharide biosynthesis. These peptide fragments, however, inhibited the action of intact acyl carrier protein in the enzymatic reaction. This suggests a role for the loop regions of the E. coli acyl carrier protein and the need for at least two regions of the protein for participation in the glucosyltransferase reaction. We have purified acyl carrier protein from eight species of Proteobacteria (including representatives from all four subgroups) and characterized the proteins as active or inhibitory in the membrane glucosyltransferase reaction. The complete or partial amino acid sequences of these acyl carrier proteins were determined. The results of site-directed mutagenesis to change amino acids conserved in active, and altered in inactive, acyl carrier proteins suggest the importance of residues Glu-4, Gln-14, Glu-21, and Asp-51. The first 3 of these residues define a face of acyl carrier protein that includes the beginning of the loop region, residues 16 to 36. Additionally, screening for membrane glucosyltransferase activity in membranes from bacterial species that had acyl carrier proteins that were active with E. coli membranes revealed the presence of glucosyltransferase activity only in the species most closely related to E. coli. Thus, it seems likely that only bacteria from the Proteobacteria subgroup gamma-3 have periplasmic glucans synthesized by the mechanism found in E. coli.

UTP: alpha-D-glucose-1-phosphate uridylyltransferase of Escherichia coli: isolation and DNA sequence of the galU gene and purification of the enzyme.

The galU gene of Escherichia coli, thought to encode the enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase, had previously been mapped to the 27-min region of the chromosome (J. A. Shapiro, J. Bacteriol. 92:518-520, 1966). By complementation of the membrane-derived oligosaccharide biosynthetic defect of strains with a galU mutation, we have now identified a plasmid containing the galU gene and have determined the nucleotide sequence of this gene. The galU gene is located immediately downstream of the hns gene, and its open reading frame would be transcribed in the direction opposite that of the hns gene (i.e., clockwise on the E. coli chromosome). The nucleotide sequences of five galU mutations were also determined. The enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase was purified from a strain containing the galU gene on a multicopy plasmid. The amino-terminal amino acid sequence (10 residues) of the purified enzyme was identical to the predicted amino acid sequence (after the initiating methionine) of the galU-encoded open reading frame. The functional enzyme appears to be a tetramer of the galU gene product.

Isolation and characterization of Escherichia coli mutants blocked in production of membrane-derived oligosaccharides.

We report a new procedure for the facile selection of mutants of Escherichia coli that are blocked in the production of membrane-derived oligosaccharides. Four phenotypic classes were identified, including two with a novel array of characteristics. The mutations mapped to two genetic loci. Mutations in the mdoA region near 23 min are in two distinct genes, only one of which is needed for the membrane-localized glucosyltransferase that catalyzes the synthesis of the beta-1,2-glucan backbone of membrane-derived oligosaccharides. Another set of mutations mapped near 27 min closely linked to osmZ; these appear to be in the galU gene.

Mechanisms of regulation of the biosynthesis of membrane-derived oligosaccharides in Escherichia coli.

The periplasmic glucans of Gram-negative bacteria, including the membrane-derived oligosaccharides (MDO) of Escherichia coli and the cyclic glucans of the Rhizobiaceae, have important but poorly understood functions in osmotic adaptation and, in the case of the Rhizobiaceae, in the complex cell-signaling of these bacteria with specific plant hosts. Experiments on the mechanisms of osmotic regulation of the biosynthesis of MDO in E. coli reported here support a model in which osmotic regulation occurs principally at the level of modulation of enzyme activity rather than at the level of gene expression. 1) Activity of the membrane-bound glucosyltransferase thought to catalyze the first and rate-making step in the biosynthesis of MDO is not altered by the osmolarity of the medium in which cells are grown. 2) Upon dilution of cells growing at high osmolarity into a medium of low osmolarity, the increased synthesis of MDO begins at maximum rate without detectable lag. 3) The activity of the membrane glucosyltransferase in vitro is strongly inhibited by high levels of salts, consistent with the view that synthesis in vivo is regulated chiefly by this mechanism, rather than by regulation of the synthesis of biosynthetic enzymes. We also find that the biosynthesis of MDO is regulated not only osmotically but also by strong feedback inhibition in response to the levels of MDO in the periplasm.

Biosynthesis and excretion of cyclic glucans by Rhizobium meliloti 1021.

Cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species play an important role in the interaction of these bacteria with plant hosts. In this study, we show that (i) the neutral cyclic glucans are the biosynthetic precursors of anionic cyclic glucans; (ii) the conversion of neutral to anionic glucans is much more rapid and more extensive in exponentially growing cultures than in cultures in the stationary phase, although the latter synthesize large amounts of glucan; and (iii) the excretion of glucan, as well as the total amount synthesized, is strongly influenced by the medium.

Biosynthesis of membrane-derived oligosaccharides. Membrane-bound glucosyltransferase system from Escherichia coli requires polyprenyl phosphate.

The periplasmic glucans of Gram-negative bacteria, including the membrane-derived oligosaccharides (MDO) of Escherichia coli and the cyclic glucans of the Rhizobiaceae, are now recognized to be a family of closely related substances with important functions in osmotic adaptation and cell signaling. The synthesis of the beta-1,2-glucan backbone of MDO is catalyzed by a membrane-bound glucosyltransferase system previously shown to require UDP-glucose and (surprisingly) acyl carrier protein (Therisod, H., Weissborn, A. C., and Kennedy, E. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7236-7240). In the present study, no glucan intermediates bound to acyl carrier protein or to UDP could detected. The enzyme system, however, was found to be strongly inhibited by bacitracin and by amphomycin. Because the two antibiotics function by forming specific complexes with polyprenyl phosphates, their inhibitory effect suggests a prenol requirement for MDO biosynthesis. Furthermore, the activity of the glucosyltransferase was greatly stimulated by the addition of polyprenyl phosphates such as decaprenyl-P and dihydroheptaprenyl-P, but not by farnesyl-P. The same membrane preparations carry out the synthesis of polyprenyl-P-glucose, which is also stimulated by added polyprenyl-P, including farnesyl-P, the most active of those tested. Pulse chase experiments, however, indicate that the endogenous pool of polyprenyl-P-glucose cannot be an obligate intermediate in the MDO glucosyltransferase system.

Cyclic glucans produced by Agrobacterium tumefaciens are substituted with sn-1-phosphoglycerol residues.

In a previous study (Miller, K.J., Kennedy, E.P. and Reinhold, V.N. (1986) Science 231, 48-51) it was reported that the biosynthesis of periplasmic cyclic beta-1,2-glucans by Agrobacterium tumefaciens is strictly osmoregulated in a pattern closely similar to that found for the membrane-derived oligosaccharides of Escherichia coli (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. USA 79, 1092-1095). In addition to the well-characterized neutral cyclic glucan, the periplasmic glucans were found to contain an anionic component not previously reported. Biosynthesis of the anionic component is osmotically regulated in a manner indistinguishable from that of the neutral cyclic beta-1,2-glucan. We now find that the anionic component consists of cyclic beta-1,2-glucans substituted with one or more sn-1-phosphoglycerol residues. The presence of sn-1-phosphoglycerol residues represents an additional, striking similarity to the membrane-derived oligosaccharides of E. coli.

An essential function for acyl carrier protein in the biosynthesis of membrane-derived oligosaccharides of Escherichia coli.

Membrane-derived oligosaccharides are branched, substituted beta-glucans localized in the periplasmic space of Escherichia coli and other Gram-negative bacteria. The biosynthesis of membrane-derived oligosaccharides and of analogous periplasmic oligosaccharides found in plant bacteria is of particular interest because it is subject to strict osmotic regulation [Miller, K.J., Kennedy, E.P., and Reinhold, V.N. (1986) Science 231, 48-51]. An enzyme system catalyzing the synthesis of the (beta 1-2)-linked glucan backbone of E. coli membrane-derived oligosaccharides from UDP-glucose requires both a membrane component and a cytosolic protein termed transglucosylation factor. The factor has now been purified to apparent homogeneity and has been found to be identical to acyl carrier protein (ACP), the phosphopantetheine-containing protein of low molecular weight that has long been known to be essential for fatty acid synthesis in E. coli and other organisms. Both are small, heat-stable, highly anionic proteins with identical chromatographic and electrophoretic behavior. ACP of the highest purity has an activity in the transglucosylation system indistinguishable from that of the protein independently purified as transglucosylation factor. Antibody raised against pure ACP completely inhibits transglucosylation activity; this inhibition is overcome by titration of the antibody with either ACP or transglucosylation factor. These findings provide evidence for an essential function of ACP unrelated to the biosynthesis of lipid.

Biosynthesis of membrane-derived oligosaccharides. Novel glucosyltransferase system from Escherichia coli for the elongation of beta 1----2-linked polyglucose chains.

Membrane-derived oligosaccharides (MDO) of Escherichia coli are a family of substituted branched oligomers containing 8-12 residues of glucose that are joined by beta 1----2 and beta 1----6 linkages. MDO are localized in the periplasmic space of the cell, and their biosynthesis is regulated by the osmolarity of the medium (Kennedy, E. P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1092-1095). We report here the initial characterization of a novel glucosyltransferase system that catalyzes the elongation of beta 1----2-linked polyglucose chains. The system requires: 1) a beta-D-glucoside such as the disaccharide sophorose (2-O-beta-D-glucosyl-glucose) or octyl beta-D-glucoside; 2) a trypsin-sensitive membrane fraction; 3) a heat-stable protein from the soluble fraction; 4) UDP-glucose; and 5) Mg2+ ions. Oligomers containing 6-10 glucose units (about the same size as MDO) that are joined by beta 1----2 linkages are major products of the enzyme system. Mutants in the recently mapped mdoA locus (Bohin, J. -P., and Kennedy, E. P. (1984) J. Bacteriol. 157, 956-957) are blocked in vivo at an early stage of MDO synthesis. It has now been found that mdoA mutants are defective in the membrane component, but not in the heat-stable protein that is required for the in vitro synthesis of beta 1----2-linked glucosyl oligomers. We conclude that the glucosyltransferase system described here has an essential function in the synthesis of MDO in vivo.