The Melibiose Carrier of Escherichia coli: Cysteine Substitutions for Individual Residues in Helix XI

The melibiose carrier from Escherichia coli is a sugar-cation cotransport system. Previously evidence was obtained that this integral membrane protein consists of 12 transmembrane helices. Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 374–396, which includes all of the residues in the proposed helix XI. The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloromercuribenzenesulfonic acid (PCMBS). Studies were carried out on both intact cells and inside out vesicles. Cysteine substitution caused loss of transport activity in seven of the mutants (K377C, G379C, A383C, F385C, L391C, G395C and Y396C). PCMBS produced more than 50% inhibition in six of the mutants (S380C, A381C, A384C, F387C, A388C and L391C). Preincubation of the cells with melibiose protected five of these residues from the inhibitory action of PCMBS. It was concluded that the residues whose cysteine derivatives were inhibited by PCMBS probably faced the aqueous channel. Received: 30 September 1999/Revised: 22 November 1999     

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. Received: 6 March 2001/Revised: 14 May 2001     

Lactose carrier mutants of Escherichia coli with changes in sugar recognition (lactose versus melibiose).

The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coli. The lactose carrier transports the alpha-galactoside sugar melibiose as well as the beta-galactoside sugar lactose. Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside beta-thio-o-nitrophenylgalactoside. Mutants that grew on melibiose minimal plates but failed to grow on lactose minimal plates were picked. In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of melibiose downhill transport. Accumulation (uphill) of melibiose was completely defective in all of the mutants. Kinetic analysis of melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for melibiose. PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method. The following six mutations were found in the lacY structural genes of individual mutants: Tyr-26-->Asp, Phe-27-->Tyr, Phe-29-->Leu, Asp-240-->Val, Leu-321-->Gln, and His-322-->Tyr. We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp-240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E. coli.     

Sugar recognition mutants of the melibiose carrier of Escherichia coli: possible structural information concerning the arrangement of membrane-bound helices and sugar/cation recognition site

Melibiose carrier mutants, isolated by growing cells on melibiose plus the non-metabolizable competitive inhibitor thiomethyl-beta-galactoside (TMG), were studied to determine sugar and cation recognition abnormalities. Most of the mutants show good transport of melibiose but have lost the recognition of TMG. In addition, most mutants show little or no transport of lactose. Cation recognition is also affected as all of these mutants have lost the ability to transport protons with melibiose. The amino acids causing these mutations were determined by sequencing the melB gene on the plasmid. The mutations were located on helices I, IV, VII, X and XI. We propose that these five helices are in proximity with each other and that they line the sugar/cation transport channel.     

Physiological evidence for an interaction between helix XI and helices I, II, and V in the melibiose carrier of Escherichia coli

In a previous study 23 residues in helix XI of the cysteine-less melibiose carrier were changed individually to cysteine. Several of these cysteine mutants (K377C, A383C, F385C, L391C, G395C) had low transport activity and they were white on melibiose MacConkey fermentation plates. After several days of incubation of these white clones on melibiose MacConkey plates a rare red mutant appeared. The plasmid DNA was then isolated and sequenced. The two second site revertants from K377C were I22S and D59A. This change of aspartic acid to a neutral residue suggests that physiologically there is an interaction between K377 and D59 (possibly a salt bridge). The revertants from A383C were in positions 20 (F20L) and 22 (I22S and I22N). Revertants of F385C were intrahelical changes (I387M and A388G) and a change in C-terminal loop (R441C). Revertants of L391C were in helix I (I22N, I22T and D19E) and helix V (A152S). Revertants of G395C were in helix I (D19E and I22N). We suggest that there is an interaction between helix XI and helices I, II, and V and proximity between these helices.     

Identification of a Region Critically Involved in the Interaction of Phlorizin with the Rabbit Sodium-D-Glucose Cotransporter SGLT1

In order to define potential interaction sites of SGLT1 with the transport inhibitor phlorizin, mutagenesis studies were performed in a hydrophobic region of loop 13 (aa 604–610), located extracellularly, close to the C-terminus. COS 7 cells were transiently transfected with the mutants and the kinetic parameters of α-methyl-d-glucopyranoside (AMG) uptake into the cells were investigated. Replacement of the respective amino acids with lysine reduced the maximal uptake rate: Y604K showed 2.2%, L606K 48.4%, F607K 15.1%, C608K 13.1%, G609K 14.1%, and L610K 17.2% of control. In all mutants the apparent K _i for phlorizin increased at least by a factor of 5 compared to the wild-type K _i of 4.6 ± 0.7 μmol/l; most striking changes were observed for Y604K (K _i = 75.3 ± 19.0 μmol/l) and C608K (K _i = 83.6 ± 13.9 μmol/l). Replacement of these amino acids with a nonpolar amino acid instead of lysine such as in Y604F, Y604G and C608A showed markedly higher affinities for phlorizin. In cells expressing the mutants the apparent affinity of AMG uptake for the sugar was not statistically different from that of the wild type (K _m = 0.8 ± 0.2 mmol/l). These studies suggest that the region between amino acids 604 and 610 is involved in the interaction between SGLT1 and phlorizin, probably by providing a hydrophobic pocket for one of the aromatic rings of the aglucone moiety of the glycoside. Received: 29 March 2001/Revised: 15 June 2001     

Energy from Redox Disproportionation of Sugar Carbon Drives Biotic and Abiotic Synthesis

To identify the energy source that drives the biosynthesis of amino acids, lipids, and nucleotides from glucose, we calculated the free energy change due to redox disproportionation of the substrate carbon of (1) 26-carbon fermentation reactions and (2) the biosynthesis of amino acids and lipids of E. coli from glucose. The free energy (cal/mmol of carbon) of these reactions was plotted as a function of the degree of redox disproportionation of carbon (disproportionative electron transfers (mmol)/mmol of carbon). The zero intercept and proportionality between energy yield and degree of redox disporportionation exhibited by this plot demonstrate that redox disproportionation is the principal energy source of these redox reactions (slope of linear fit =−10.4 cal/mmol of disproportionative electron transfers). The energy and disproportionation values of E. coli amino acid and lipid biosynthesis from glucose lie near this linear curve fit with redox disproportionation accounting for 84% and 96% (and ATP only 6% and 1%) of the total energy of amino acid and lipid biosynthesis, respectively. These observations establish that redox disproportionation of carbon, and not ATP, is the primary energy source driving amino acid and lipid biosynthesis from glucose. In contrast, we found that nucleotide biosynthesis involves very little redox disproportionation, and consequently depends almost entirely on ATP for energy. The function of sugar redox disproportionation as the major source of free energy for the biosynthesis of amino acids and lipids suggests that sugar disproportionation played a central role in the origin of metabolism, and probably the origin of life. Received: 18 April 1996 / Accepted: 31 October 1996     

Physiological evidence for an interaction between helices II and XI in the melibiose carrier of Escherichia coli

The melibiose carrier from Escherichia coli is a cation-substrate cotransporter that catalyzes the accumulation of galactosides at the expense of H(+), Na(+), or Li(+) electrochemical gradients. Charged residues on transmembrane domains in the amino-terminal portion of this carrier play an important role in the recognition of cations, while the carboxyl portion of the protein seems to be important for sugar recognition. In the present study, we substituted Lys-377 on helix XI with Val. This mutant carrier, K377V, had reduced melibiose transport activity. We subsequently used this mutant for the isolation of functional second-site revertants. Revertant strains showed the additional substitutions of Val or Asn for Asp-59 (helix II), or Leu for Phe-20 (helix I). Isolation of revertant strains where both Lys-377 and Asp-59 are substituted with neutral residues suggested the possibility that a salt bridge exists between helix II and helix XI. To further test this idea, we constructed three additional site-directed mutants: Asp-59-->Lys (D59K), Lys-377-->Asp (K377D), and a double mutant, Asp-59-->Lys/Lys-377-->Asp (D59K/K377D), in which the position of these charges was exchanged. K377D accumulated melibiose only marginally while D59K could not accumulate. However, the D59K/K377D double mutant accumulated melibiose to a modest level although this activity was no longer stimulated by Na(+). We suggest that Asp-59 and Lys-377 interact via a salt bridge that brings helix II and helix XI close to one another in the three-dimensional structure of the carrier.     

Altered sugar selection and transport conferred by spontaneous point and deletion mutations in the lactose carrier of Escherichia coli

Spontaneous mutants harboring the lacY gene on an F'-factor were isolated. Those mutants that failed to grow on 5 mM lactose minimal media plates were chosen for further study. The mutants showed striking mutations in the lactose carrier as well as in sugar selection properties during transport assays. DNA sequencing of the lacY gene of the mutants revealed the following mutations: M-1-I, R-144-W, G-370-C and a deletion of residues 387-392, located in helix 12 of the carrier. Transport studies indicated that ONPG transport ranged between 8 and 25% of normal for the M-1-I, G-370-C and D387-392 mutants and 51% of normal for the R-144-W mutant. The downhill transport of lactose was 2-fold greater than for melibiose in cells harboring the M-1-I mutation and 3-fold higher for cells with the G-370-C mutation. On the other hand, cells with the D387-392-deletion mutation showed no lactose downhill transport, but 47% melibiose transport. Accumulation of TMG, a lactose analog, was 3-fold higher than the accumulation of melibiose in cells with the G-370-C mutation. On the other hand, in cells with the D387-392 mutation, TMG accumulation was completely defective, whereas melibiose accumulation was 50-fold higher than that of TMG, indicating that one or more of these residues in helix 12 of the carrier play a role in the active transport of b-galactoside, but not a-galactoside sugars. Initial lactose downhill transport rates were too unreliable to obtain trustworthy kinetic data. TMG and melibiose accumulation activities were present, but severely reduced in the mutant containing the R144W mutation, confirming that Arg-144 is important for active transport. All transport data were normalized for expression levels. The results indicate that the affected residues play a role in dictating sugar specificity and transport in the lactose carrier. The results here are novel in that they represent mutations in unique locations along the lactose carrier protein. For example, the M-1-I mutation was located at the N-terminal cytoplasmic tail of the carrier. Furthermore, G-370-C was located in the periplasmic loop between helices 11 and 12, suggesting a role for residues in this loop in mediating sugar selection.     

The Conserved Motif in Hydrophilic Loop 2/3 and Loop 8/9 of the Lactose Permease of Escherichia coli. Analysis of Suppressor Mutations

The major facilitator superfamily (MFS) of transport proteins, which includes the lactose permease of Escherichia coli, contains a conserved motif G-X-X-X-D/E-R/K-X-G-R/K-R/K in the loops that connect transmembrane segments 2 and 3, and transmembrane segments 8 and 9. In three previous studies (Jessen-Marshall, A.E., & Brooker, R.J. 1996. J. Biol. Chem. 271:1400–1404; Jessen-Marshall, A.E., Parker, N., & Brooker, R.J. 1997. J. Bacteriol. 179:2616–2622; and Pazdernik, N., Cain, S.M., & Brooker, R.J. 1997. J. Biol. Chem. 272:26110–26116), suppressor mutations at twenty different sites were identified which restore function to mutant permeases that have deleterious mutations in the conserved loop 2/3 or loop 8/9 motif. In the current study, several of these second-site suppressor mutations have been separated from the original mutation in the conserved motif. The loop 2/3 suppressors were then coupled to a loop 8/9 mutation (P280L) and the loop 8/9 suppressors were coupled to a loop 2/3 mutation (i.e., G64S) to determine if the suppressors could restore function only to a loop 2/3 mutation, a loop 8/9 mutation, or both. The single parent mutations changing the first position in loop 2/3 (i.e., G64S) and loop 8/9 (i.e., P280L) had less than 4% lactose transport activity. Interestingly, most of the suppressors were very inhibitory when separated from the parent mutation. Two suppressors, A50T and G370V, restored substantial transport activity when individually coupled to the mutation in loop 2/3 and also when coupled to the corresponding mutation in loop 8/9. In other words, these suppressors could alleviate a defect imposed by mutations in either half of the permease. From a kinetic analysis, these suppressors were shown to exert their effects by increasing the V _max values for lactose transport compared with the single G64S and P280L strains. These results are discussed within the context of our model in which the two halves of the lactose permease interact at a rotationally symmetrical interface, and that lactose transport is mediated by conformational changes at the interface. Received: 18 November 1999/Revised: 11 April 2000     

Suppression of Inward-Rectifying K+ Channels KAT1 and AKT2 by Dominant Negative Point Mutations in the KAT1 α-Subunit

The Arabidopsis thaliana cDNA, KAT1 encodes a hyperpolarization-activated K⁺ (K⁺ _in ) channel. In the present study, we identify and characterize dominant negative point mutations that suppress K⁺ _in channel function. Effects of two mutations located in the H5 region of KAT1, at positions 256 (T256R) and 262 (G262K), were studied. The co-expression of either T256R or G262K mutants with KAT1 produced an inhibition of K⁺ currents upon membrane hyperpolarization. The magnitude of this inhibition was dependent upon the molar ratio of cRNA for wild-type to mutant channel subunits injected. Inhibition of KAT1 currents by the co-expression of T256R or G262K did not greatly affect the ion selectivity of residual currents for Rb⁺, Na⁺, Li⁺, or Cs⁺. When T256R or G262K were co-expressed with a different K⁺ channel, AKT2, an inhibition of the channel currents was also observed. Voltage-dependent Cs⁺ block experiments with co-expressed wild type, KAT1 and AKT2, channels further indicated that KAT1 and AKT2 formed heteromultimers. These data show that AKT2 and KAT1 are able to co-assemble and suggest that suppression of channel function can be pursued in vivo by the expression of the dominant negative K ⁺ _in channel mutants described here. Received: 2 July 1998/Revised: 23 October 1998     

A Kinetic Model with Ordered Cytoplasmic Dissociation for SUC1, an Arabidopsis H+/Sucrose Cotransporter Expressed in Xenopus Oocytes

To elucidate the kinetic properties of the Arabidopsis H⁺/sucrose cotransporter, SUC1, with respect to transmembrane voltage and ligand concentrations, the transport system was heterologously expressed in Xenopus laevis oocytes. Steady-state plasma membrane currents associated with transport of sucrose were measured with two-electrode voltage clamp over the voltage range −180 to +40 mV as a function of extracellular pH and sugar concentrations. At any given voltage, currents exhibited hyperbolic kinetics with respect to extracellular H⁺ and sugar concentrations, and this enabled determination of values for the maximum currents in the presence of each ligand (i ^H _max , i ^S _max for H⁺ and sucrose) and of the ligand concentrations eliciting half-maximal currents (K ^H _m , K ^S _m ). The i ^H _max and i ^S _max exhibited marked and statistically significant increases as a function of increasingly negative membrane potential. However, the K ^H _m and K ^S _m decreased with increasingly negative membrane potential. Furthermore, at any given voltage, i ^S _max increased and K ^S _m decreased as a function of the external H⁺ concentration. Eight six-state carrier models—which comprised the four possible permutations of intracellular and extracellular ligand binding order, each with charge translocation on the sugar-loaded or -unloaded forms of the carrier—were analyzed algebraically with respect to their competence to account for the ensemble of kinetic observations. Of these, two models (first-on, first-off and last-on, first-off with respect to sucrose binding as it passes from outside to inside the cell and with charge translocation on the loaded form of the carrier) exhibit sufficient kinetic flexibility to describe the observations. Combining these two, a single model emerges in which the binding on the external side can be random, but it can only be ordered on the inside, with the sugar dissociating before the proton. Received: 23 January 1996/Revised: 16 April 1997     

Slow Gating of Gap Junction Channels and Calmodulin

Certain COOH-terminus mutants of connexin32 (Cx32) were previously shown to form channels with unusual transjuctional voltage (V _j ) sensitivity when tested heterotypically in oocytes against Cx32 wild type. Junctional conductance (G _j ) slowly increased by severalfold or decreases to nearly zero with V _j positive or negative, respectively, at mutant side, and V _j positive at mutant side reversed CO₂-induced uncoupling. This suggested that the CO₂-sensitive gate might be a V _j -sensitive slow gate. Based on previous data for calmodulin (CaM) involvement in gap junction function, we have hypothesized that the slow gate could be a CaM-like pore plugging molecule (cork gating model). This study describes a similar behavior in heterotypic channels between Cx32 and each of four new Cx32 mutants modified in cytoplasmic-loop and/or COOH-terminus residues. The mutants are: ML/NN+3R/N, 3R/N, ML/NN and ML/EE; in these mutants, N or E replace M105 and L106, and N replace R215, R219 and R220. This study also reports that inhibition of CaM expression strongly reduces V _j and CO₂ sensitivities of two of the most effective mutants, suggesting a CaM role in slow and chemical gating. Received: 19 April 2000/Revised: 11 August 2000     

Sugar Transport Heterogeneity in the Kidney: Two Independent Transporters or Different Transport Modes through an Oligomeric Protein? 1. Glucose Transport Studies

The kinetics of Na⁺/d-glucose cotransport (SGLT) were reevaluated in rabbit renal brush border membrane vesicles isolated from the whole kidney cortex using a fast-sampling, rapid-filtration apparatus (FSRFA, US patent #5,330,717) for uptake measurements. Our results confirm SGLT heterogeneity in this preparation, and both high (HAG) and low (LAG) affinity glucose transport pathways can be separated over the 15–30°C range of temperatures. It is further shown that: (i) Na⁺ is an essential activator of both HAG and LAG; (ii) similar energies of activation can be estimated from the linear Arrhenius plots constructed from the V _max data of HAG and LAG, thus suggesting that the lipid composition and/or the physical state of the membrane do not affect much the functioning of SGLT; (iii) similar V _max values are observed for glucose and galactose transport through HAG and LAG, thus demonstrating that the two substrates share the same carrier agencies; and (iv) phlorizin inhibits both HAG and LAG competitively and with equal potency (K _i = 15 μm). Individually, these data do not allow us to resolve conclusively whether the kinetic heterogeneity of SGLT results from the expression in the proximal tubule of either two independent transporters (rSGLT1 and rSGLT2) or from a unique transporter (rSGLT1) showing allosteric kinetics. Altogether and compared to the kinetic characteristics of the cloned SGLT1 and SGLT2 systems, they do point to a number of inconsistencies that lead us to conclude the latter possibility, although it is recognized that the two alternatives are not mutually exclusive. It is further suggested, from the differences in the K _m values of HAG transport in the kidney as compared to the small intestine and SGLT1 cRNA-injected oocytes, that renal SGLT1 activity is somehow modulated, maybe through heteroassociation with (a) regulatory subunit(s) that might also contribute quite significantly to sugar transport heterogeneity in the kidney proximal tubule. Received: 25 October 1995/Revised: 10 June 1996     

Limitations of compositional approach to identifying horizontally transferred genes

Genes with atypical G+C content and pattern of codon usage in a certain genome are possibly of exotic origin, and this idea has been applied to identify horizontal events. In this way, it was postulated that a total of 755 genes in the E. coli genome are relics of horizontal events after the divergence of E. coli from the Salmonella lineage 100 million years ago (Lawrence and Ochman, 1998). In this paper we propose a new way to study sequence composition more thoroughly. We found that although the 755 genes differ in composition from other genes in the E. coli genome, the difference is minor. If we accepted that these genes are horizontally transferred, then (1) it would be more likely that they were transferred from genomes evolutionarily closely related to E. coli; but (2) the dating method used by Lawrence and Ochman (1997, 1998) largely underestimated the average age of introduced sequences in the E. coli genome, in particular, most of the 755 genes should be introduced into E. coli before, instead of after, the divergence of E. coli from the Salmonella lineage. Our study reveals that atypical G+C content and pattern of codon usage are not reliable indicators of horizontal gene transfer events. Received: 27 September 2000 / Accepted: 9 April 2001     

Cation-Sugar Cotransport in the Melibiose Transport System of Escherichia coli

The entry of Na⁺ or H⁺ into cells of Escherichia coli via the melibiose transport system was stimulated by the addition of certain galactosides. The principal cell used in these studies (W3133) was a lactose transport negative strain of E. coli possessing an inducible melibiose transport system. Such cells were grown in the presence of melibiose, washed, and incubated in the presence of 25 μM Na⁺. The addition of thiomethylgalactoside (TMG) resulted in a fall in Na⁺ concentration in the incubation medium. No TMG-stimulated Na⁺ movement was observed in uninduced cells. In an α-galactosidase negative derivative of W3133 (RA11) a sugar-stimulated Na⁺ uptake was observed in meliboise-induced cells on the addition of melibiose, thiodigalactoside, methyl-α-galactoside, methyl-β-galactoside, and galactose, but not lactose. It was inferred from these studies that the substrates of the melibiose system enter the cell on the melibiose carrier associated with the simultaneous entry of Na⁺ when this cation is present in the incubation medium.

Extracellular pH was measured in unbuffered suspensions of induced cells in order to study proton movement across the membrane of cells exposed to different galactosides. In the absence of external Na⁺ or Li⁺ the addition of melibiose or methyl-α-galactoside resulted in marked alkalinization of the external medium (consistent with H⁺-sugar cotransport). On the other hand TMG, thiodigalactoside, and methyl-β-galactoside gave no proton movement under these conditions. When Na⁺ was present, the addition of TMG or melibiose resulted in acidification of the medium. This observation is consistent with the view that the entry of Na⁺ with TMG or melibiose carries into the cell a positive charge (Na⁺) which provides the driving force for the diffusion of protons out of the cell. It is concluded that the melibiose carrier recognition of cations differs with different substrates.     

A Triple Mutant, K319N/H322Q/E325Q, of the Lactose Permease Cotransports H+ with Thiodigalactoside

In a previous study, we characterized a lactose permease mutant (K319N/E325Q) that can transport H⁺ ions with sugar. This result was surprising because other studies had suggested that Glu-325 plays an essential role in H⁺ binding. To determine if the lactose permease contains one or more auxiliary H⁺ binding sites, we began with the K319N/E325Q strain, which catalyzes a sugar-dependent H⁺ leak, and isolated third site suppressor mutations that blocked the H⁺ leak. Three types of suppressors were obtained: H322Y, H322R, and M299I. These mutations blocked the H⁺ leak and elevated the apparent K _m value for lactose. The M299I and H322Y suppressors could still transport H⁺ with β-d-thiodigalactoside (TDG), but the H322R strain appeared uncoupled for H⁺/sugar cotransport. Four mutant strains containing a nonionizable substitution at codon 322 (H322Q) were analyzed. None of these were able to catalyze uphill accumulation of lactose, however, all showed some level of substrate-induced proton accumulation. The level seemed to vary based on the substrate being analyzed (lactose or TDG). Most interestingly, a triple mutant, K319N/H322Q/E325Q, catalyzed robust H⁺ transport with TDG. These novel results suggest an alternative mechanism of lactose permease cation binding and transport, possibly involving hydronium ion (H₃O⁺). Received: 6 November 2000/Revised: 23 March 2001     

The Interaction of Protein Structure, Selection, and Recombination on the Evolution of the Type-1 Fimbrial Major Subunit (fimA) from Escherichia coli

Fimbrial adhesins allow bacteria to interact with and attach to their environment. The bacteria possibly benefit from these interactions, but all external structures including adhesins also allow bacteria to be identified by other organisms. Thus adhesion molecules might be under multiple forms of selection including selection to constrain functional interactions or evolve novel epitopes to avoid recognition. We address these issues by studying genetic diversity in the Escherichia coli type-1 fimbrial major subunit, fimA. Overall, sequence diversity in fimA is high (π= 0.07) relative to that in other E. coli genes. High diversity is a function of positive diversifying selection, as detected by d _N/d _S ratios higher than 1.0, and amino acid residuces subject to diversifying selection are nonrandomly clustered on the exterior surface of the peptide. In addition, McDonald and Kreitman tests suggest that there has been historical but not current directional selection at fimA between E. coli and Salmonella. Finally, some regions of the fimA peptide appear to be under strong structural constraint within E. coli, particularly the interior regions of the molecule that is involved in subunit to subunit interaction. Recombination also plays a major role contributing to E. coli fimA allelic variation and estimates of recombination (2N _e c) and mutation (2N _eμ) are about the same. Recombination may act to separate the diverse evolutionary forces in different regions of the fimA peptide. Received: 13 April 2000 / Accepted: 28 October 2000     

Altered substrate selection of the melibiose transporter (MelY) of Enterobacter cloacae involving point mutations in Leu-88, Leu-91, and Ala-182 that confer enhanced maltose transport

We isolated mutants of Escherichia coli HS4006 containing the melibiose-H(+) symporter (MelY) from Enterobacter cloacae that had enhanced fermentation on 1% maltose MacConkey plates. DNA sequencing revealed three site classes of mutations: L-88-P, L-91-P, and A-182-P. The mutants L-88-P and L-91-P had 3.6- and 5.1-fold greater maltose uptake than the wild type and enhanced apparent affinities for maltose. Energy-coupled transport was defective for melibiose accumulation, but detectable maltose accumulation for the mutants indicated that active transport is dependent upon the substrate transported through the carrier. We conclude that the residues Leu-88, Leu-91 (transmembrane segment 3 [TMS-3]), and Ala-182 (TMS-6) of MelY mediate sugar selection. These data represent the first MelY mutations that confer changes in sugar selection.     

Proton-linked sugar transport systems in bacteria

The cell membranes of various bacteria contain proton-linked transport systems ford-xylose,l-arabinose,d-galactose,d-glucose,l-rhamnose,l-fucose, lactose, and melibiose. The melibiose transporter ofE. coli is linked to both Na⁺ and H⁺ translocation. The substrate and inhibitor specificities of the monosaccharide transporters are described. By locating, cloning, and sequencing the genes encoding the sugar/H⁺ transporters inE. coli, the primary sequences of the transport proteins have been deduced. Those for xylose/H⁺, arabinose/H⁺, and galactose/H⁺ transport are homologous to each other. Furthermore, they are just as similar to the primary sequences of the following: glucose transport proteins found in a Cyanobacterium, yeast, alga, rat, mouse, and man; proteins for transport of galactose, lactose, or maltose in species of yeast; and to a developmentally regulated protein of Leishmania for which a function is not yet established. Some of these proteins catalyze facilitated diffusion of the sugar without cation transport. From the alignments of the homologous amino acid sequences, predictions of common structural features can be made: there are likely to be twelve membrane-spanning -helices, possibly in two groups of six, there is a central hydrophilic region, probably comprised largely of -helix; the highly conserved amino acid residues (40–50 out of 472–522 total) form discrete patterns or motifs throughout the proteins that are presumably critical for substrate recognition and the molecular mechanism of transport. Some of these features are found also in other transport proteins for citrate, tetracycline, lactose, or melibiose, the primary sequences of which are not similar to each other or to the homologous series of transporters. The glucose/Na⁺ transporter of rabbit and man is different in primary sequence to all the other sugar transporters characterized, but it is homologous to the proline/Na⁺ transporter ofE. coli, and there is evidence for its structural similarity to glucose/H⁺ transporters in Plants.In vivo andin vitro mutagenesis of the lactose/H⁺ and melibiose/Na⁺ (H⁺) transporters ofE. coli has identified individual amino acid residues alterations of which affect sugar and/or cation recognition and parameters of transport. Most of the bacterial transport proteins have been identified and the lactose/H⁺ transporter has been purified. The directions of future investigations are discussed.