Subunit and amino acid interactions in the Escherichia coli mannitol permease: a functional complementation study of coexpressed mutant permease proteins.

Mannitol-specific enzyme II, or mannitol permease, of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system of Escherichia coli carries out the transport and phosphorylation of D-mannitol and is most active as a dimer in the membrane. We recently reported the importance of a glutamate residue at position 257 in the binding and transport of mannitol by this protein (C. Saraceni-Richards and G. R. Jacobson, J. Bacteriol. 179:1135-1142, 1997). Replacing Glu-257 with alanine (E257A) or glutamine (E257Q) eliminated detectable mannitol binding and transport by the permease. In contrast, an E257D mutant protein was able to bind and phosphorylate mannitol in a manner similar to that of the wild-type protein but was severely defective in mannitol uptake. In this study, we have coexpressed proteins containing mutations at position 257 with other inactive permeases containing mutations in each of the three domains of this protein. Activities of any active heterodimers resulting from this coexpression were measured. The results show that various inactive mutant permease proteins can complement proteins containing mutations at position 257. In addition, we show that both Glu at position 257 and His at position 195, both of which are in the membrane-bound C domain of the protein, must be on the same subunit of a permease dimer in order for efficient mannitol phosphorylation and uptake to occur. The results also suggest that mannitol bound to the opposite subunit within a permease heterodimer can be phosphorylated by the subunit containing the E257A mutation (which cannot bind mannitol) and support a model in which there are separate binding sites on each subunit within a permease dimer. Finally, we provide evidence from these studies that high-affinity mannitol binding is necessary for efficient transport by mannitol permease.     

Intracellular activation of albomycin in Escherichia coli and Salmonella typhimurium.

The antibiotic albomycin is actively taken up by Escherichia coli via the transport system for the structurally similar iron complex ferrichrome. Albomycin is cleaved, and the antibiotically active moiety is released into the cytoplasm, whereas the iron carrier moiety appears in the medium. Besides transport-negative mutants, additional albomycin-resistant mutants were isolated. The mutations were mapped outside the transport genes close to the pyrD gene at 21 min. The mutants were devoid of peptidase N activity. The molecular weight, sensitivity to inhibitors, and cytoplasmic location of the enzyme hydrolyzing albomycin in vitro corresponded to the known properties of peptidase N. The aminoacyl thioribosyl pyrimidine moiety of albomycin apparently has to be cleaved off the iron chelate transport vehicle to inhibit growth. Peptidase N is the major hydrolyzing enzyme. In Salmonella typhimurium peptidase N and peptidase A were equally active in hydrolyzing and activating albomycin.     

Inactivation of membrane transport in Escherichia coli by near-ultraviolet light.

Evidence is presented that near-ultraviolet (near-UV) light can alter galactoside transport in Escherichia coli in several independent ways. It can inactivate the permease system per se, it can interfere with metabolic energy production or transfer, and it can cause an increase in the generalized permeability of the membrane. Earlier publications suggested that near-UV destroys cofactors needed for electron transport and thus places a limitation on energy reserves. In agreement, we found that the active accumulation of [14C]thiomethyl-beta-D-galactopyranoside is decreased after irradiation by a larger factor than that due to action directly on the permease system. The effect on the latter was measured by the decrease in the rate of o-nitrophenyl-beta-D-galactopyranoside (ONPG) transport. As evidence that energy supplies for this "downhill" process did not become rate limiting after irradiation, we found that carbonylcyanide-m-chlorophenyl-hydrazone did not stimulate ONPG transport of irradiated cells. Cells genetically deficient in functional permease or cells treated with formaldehyde still transport ONPG passively, although at much lower rates. With the use of such cells, it was found that high fluences (doses) made the cells leaky. Further evidence that the permease system and the metabolic energy system can be inactivated independently is also presented. It is shown that a photoproduct from the irradiation of chloramphenicol inactivates the permease system much more efficiently than the energy system. In addition, it is shown that thio-beta-D-digalactopyranoside protects the permease system, but not the energy system, both against direct inactivation by near-UV and against photosensitized inactivation in the presence of chloramphenicol.     

A di- and tripeptide transport system can supply Listeria monocytogenes Scott A with amino acids essential for growth.

Listeria monocytogenes takes up di- and tripeptides via a proton motive force-dependent carrier protein. This peptide transport system resembles the recently cloned and sequenced secondary di- and tripeptide transport system of Lactococcus lactis (A. Hagting, E. R. S. Kunji, K. J. Leenhouts, B. Poolman, and W. N. Konings, J. Biol. Chem. 269:11391-11399, 1994). The peptide permease of L. monocytogenes has a broad substrate specificity and allows transport of the nonpeptide substrate 5-aminolevulinic acid, the toxic di- and tripeptide analogs, alanyl-beta-chloroalanine and alanyl-alanyl-beta-chloroalanine, and various di- and tripeptides. No extracellular peptide hydrolysis was detected, indicating that peptides are hydrolyzed after being transported into the cell. Indeed, peptidase activities in response to various synthetic substrates were detected in cell extracts obtained from L. monocytogenes cells grown in brain heart infusion broth or defined medium. The di- and tripeptide permease can supply L. monocytogenes with essential amino acids for growth and might contribute to growth of this pathogen in various foods where peptides are supplied by proteolytic activity of other microorganisms present in these foods. Possible roles of this di- and tripeptide transport system in the osmoregulation and virulence of L. monocytogenes are discussed.     

A monocarboxylate permease of Rhizobium leguminosarum is the first member of a new subfamily of transporters

Amino acid transport by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra). However, mutation of these transporters does not prevent this organism from utilizing alanine for growth. An R. leguminosarum permease (MctP) has been identified which is required for optimal growth on alanine as a sole carbon and nitrogen source. Characterization of MctP confirmed that it transports alanine (K(m) = 0.56 mM) and other monocarboxylates such as lactate and pyruvate (K(m) = 4.4 and 3.8 micro M, respectively). Uptake inhibition studies indicate that propionate, butyrate, alpha-hydroxybutyrate, and acetate are also transported by MctP, with the apparent affinity for solutes demonstrating a preference for C3-monocarboxylates. MctP has significant sequence similarity to members of the sodium/solute symporter family. However, sequence comparisons suggest that it is the first characterized permease of a new subfamily of transporters. While transport via MctP was inhibited by CCCP, it was not apparently affected by the concentration of sodium. In contrast, glutamate uptake in R. leguminosarum by the Escherichia coli GltS system did require sodium, which suggests that MctP may be proton coupled. Uncharacterized members of this new subfamily have been identified in a broad taxonomic range of species, including proteobacteria of the beta-subdivision, gram-positive bacteria, and archaea. A two-component sensor-regulator (MctSR), encoded by genes adjacent to mctP, is required for activation of mctP expression.     

Genetic characterization and molecular cloning of the tripeptide permease (tpp) genes of Salmonella typhimurium.

Of the three bacterial peptide transport systems only one, the oligopeptide permease, has been characterized in any detail. We have now isolated Salmonella typhimurium mutants deficient in a second transport system, the tripeptide permease (Tpp), using the toxic peptide alafosfalin. Alafosfalin resistance mutations map at three loci, the gene encoding peptidase A (pepA) and two transport-defective loci, tppA and tppB. Locus tppA has been mapped to 74 min on the S. typhimurium chromosome, cotransducible with aroB, and is a positive regulator of tppB. Locus tppB maps at 27 min in the cotransduction gap between purB and pyrF. We cloned tppB, the structural locus for the tripeptide permease. Two simple methods are described for mapping the location of cloned DNA fragments on the chromosome of S. typhimurium.     

Antibacterial activity of phosphono dipeptides based on 1-amino-1-methylethanephosphonic acid

Phosphono dipeptides containing 1-amino-1-methylethanephosphonic acid (phosphonic acid analogue of alpha-methylalanine, MeAlaP) and glycine, alanine, valine, leucine phenylalanine, proline, methionine or lysine as N- terminal component were synthesized in order to determine their antibacterial properties. Peptides containing alanine, leucine, valine phenylalanine and methionine showed marked in vitro activity, especially against Escherichia coli and Serratia marcescens strains. There were, however, generally less potent than the respective phosphono dipeptides based on 1-aminoethanephosphonic acid (phosphonic acid analogue of alanine, AlaP). The possible mechanism of action of the peptides of MeAlaP involves their active transport into the bacterial cell, followed by intracellular release of MeAlaP, which most likely inhibits alanine racemase, a key enzyme in peptidoglycan biosynthesis. Studies on the uptake of AlaMeAlaP and LeuMeAlaP by Escherichia coli mutants defective in the oligopeptide permease suggest that these peptides are not transported by the oligopeptide transport system.     

Ureidosuccinic acid permeation in Saccharomyces cerevisiae.

Some strains of Saccharomyces cerevisiae exhibit a specific transport system for ureidosuccinic acid, which is regulated by nitrogen metabolism. Ureidosuccinic acid uptake occurs with proline but with ammonium sulfate as nitrogen source it is inhibited. The V for transport is 20-25 mumol/ml cell water per min. The apparent Km is 3-10(-5) M. For the urep1 mutant (ureidosuccinic acid permease less) the internal concentration never exceeds the external one. In the permease plus strain ureidosuccinic acid can be concentrated up to 10 000 fold and the accumulated compound remains unchanged in the cells. Energy poisons such as dinitrophenol, carbonyl cyanide-m-chlorophenyldrazone (CCCP) or NaN3 inhibit the uptake. No significant efflux of the accumulated compound occurs even in the presence of these drugs. The specificity of the permease is very strict, only amino acids carrying an alpha-N-carbamyl group are strongly competitive inhibitors. The high concentration capacity of the cells and lack of active exit of the accumulated compound support the hypothesis of a carrier mediated active transport system.     

Product induction in Pseudomonas acidovorans of a permease system which transports L-tryptophan

Growth of Pseudomonas acidovorans in the presence of l-tryptophan resulted in the appearance of a tryptophan transport system which was extremely sensitive to sodium azide or 2,4-dinitrophenol. Asparagine-grown cells possessed no detectable tryptophan "permease" activity. Substitution of l-kynurenine for l-tryptophan in the growth medium also induced the tryptophan permease activity, along with tryptophan oxygenase and kynurenine formamidase. This is the first reported example of the product induction of a permease activity. Irrespective of whether Pseudomonas cells are grown in the presence of d- or l-tryptophan, the resulting induced tryptophan permease activity is specific for the l-isomer. In addition, the radioactive compounds l-leucine, l-phenylalanine, or dl-5-hydroxytryptophan are not transported. When dl-5-fluorotryptophan is a component of the inducing medium (with l-tryptophan), induction of tryptophan permease activity, as well as tryptophan oxygenase, is inhibited. In the permease assay system, using normally induced cells, the fluoroanalogue inhibited strikingly tryptophan transport. Therefore, this analogue may inhibit induction by blocking inducer transport into the cell. When added to the l-tryptophan-inducing medium, dl-7-azatryptophan markedly enhanced induction of tryptophan oxygenase, but the level of tryptophan permease activity was not further elevated. The mechanism of this analogue is unclear at present. Invariant tryptophan permease activity levels are found in cells grown with 5 or 15 mml-tryptophan or 5 mml-kynurenine, whereas the respective tryptophan oxygenase levels are greatly different. Together with other results, these results indicate that the synthesis of tryptophan permease activity is not coordinate with that of tryptophan oxygenase. Tryptophan transport is strongly inhibited by l-formylkynurenine and by l-kynurenine. These two metabolites were prepared in radioactive form, and they are actively transported following bacterial growth on l-tryptophan or l-kynurenine. Preliminary results suggest the tryptophan permease activity may be distinct from the permease(s) activity for l-formylkynurenine and l-kynurenine. Kynurenine, then, is capable of inducing tryptophan permease and kynurenine permease activities.     

A topological model for the general aromatic amino acid permease, AroP, of Escherichia coli.

The general aromatic amino acid permease, AroP, of Escherichia coli is responsible for the active transport of phenylalanine, tyrosine, and tryptophan. A proposed topological model for the AroP permease, consisting of 12 hydrophobic transmembrane spans connected by hydrophilic loops, is very similar to that of the closely related phenylalanine-specific permease. The validity of this model and its similarity to that of the PheP permease were investigated by studying fusion proteins of AroP permease and alkaline phosphatase. Based on the results obtained from the AroP-alkaline phosphatase sandwich fusions, we have significantly revised the proposed topological model for AroP in two regions. In this modified AroP topological model, the three charged residues E151, E153, and K160 are repositioned within the membrane in span 5. These three residues are conserved in a large family of amino acid transport proteins, and site-directed mutagenesis identifies them as being essential for transport activity. It is postulated that these residues together with E110 in transmembrane span 3 may be involved in a proton relay system.     

Characterization of Constitutive Galactose Permease Mutants in Salmonella typhimurium

Salmonella typhimurium strains, lacking both enzyme I and the phosphocarrier protein, HPr, of the phosphoenolpyruvate-sugar phosphotransferase system, cannot transport or metabolize glucose and other sugar substrates of this enzyme system. Mutants which regain the ability to specifically utilize glucose were found to constitutively synthesize a galactose permease by virtue of a mutation in the galR gene. This permease, shown to be an active transport system, does not require HPr or enzyme I for activity.     

New Plasmid System To Select for Saccharomyces cerevisiae Purine-Cytosine Permease Affinity Mutants

The FCY2 gene of Saccharomyces cerevisiae encodes a purine-cytosine permease (PCP) that mediates the active transport of purines and cytosine. A structure-function model for this PCP has been recently proposed. In this study, we developed a plasmid-based system that generated a number of affinity-mutated alleles, enabling us to define new amino acids critical for permease function.     

Reversible topological organization within a polytopic membrane protein is governed by a change in membrane phospholipid composition

Once inserted, transmembrane segments of polytopic membrane proteins are generally considered stably oriented due to the large free energy barrier to topological reorientation of adjacent extramembrane domains. However, the topology and function of the polytopic membrane protein lactose permease of Escherichia coli are dependent on the membrane phospholipid composition, revealing topological dynamics of transmembrane domains after stable membrane insertion (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107-2116). In this study, we show that the high affinity phenylalanine permease PheP shares many similarities with lactose permease. PheP assembled in a mutant of E. coli lacking phosphatidylethanolamine (PE) exhibited significantly reduced active transport function and a complete inversion in topological orientation of the N terminus and adjoining transmembrane hairpin loop compared with PheP in a PE-containing strain. Introduction of PE following the assembly of PheP triggered a reorientation of the N terminus and adjacent hairpin to their native orientation associated with regain of wild-type transport function. The reversible orientation of these secondary transport proteins in response to a change in phospholipid composition might be a result of inherent conformational flexibility necessary for transport function or during protein assembly.     

Markerless mutagenesis in Methanococcus maripaludis demonstrates roles for alanine dehydrogenase, alanine racemase, and alanine permease

Among the archaea, Methanococcus maripaludis has the unusual ability to use L- or D-alanine as a nitrogen source. To understand how this occurs, we tested the roles of three adjacent genes encoding homologs of alanine dehydrogenase, alanine racemase, and alanine permease. To produce mutations in these genes, we devised a method for markerless mutagenesis that builds on previously established genetic tools for M. maripaludis. The technique uses a negative selection strategy that takes advantage of the ability of the M. maripaludis hpt gene encoding hypoxanthine phosphoribosyltransferase to confer sensitivity to the base analog 8-azahypoxanthine. In addition, we developed a negative selection method to stably incorporate constructs into the genome at the site of the upt gene encoding uracil phosphoribosyltransferase. Mutants with in-frame deletion mutations in the genes for alanine dehydrogenase and alanine permease lost the ability to grow on either isomer of alanine, while a mutant with an in-frame deletion mutation in the gene for alanine racemase lost only the ability to grow on D-alanine. The wild-type gene for alanine dehydrogenase, incorporated into the upt site, complemented the alanine dehydrogenase mutation. Hence, the permease is required for the transport of either isomer, the dehydrogenase is specific for the L isomer, and the racemase converts the D isomer to the L isomer. Phylogenetic analysis indicated that all three genes had been acquired by lateral gene transfer from the low-moles-percent G+C gram-positive bacteria.     

Expression of the cloned uracil permease gene of Saccharomyces cerevisiae in a heterologous membrane

A piece of DNA of the yeast Saccharomyces cerevisiae complementing the uracil permease gene was introduced into a plasmid able to replicate autonomously in Schizosaccharomyces pombe. A strain of S. pombe lacking uracil transport activity was transformed with this new plasmid carrying the gene of S. cerevisiae. The behaviour of the transformant shows not only an expression of the uracil permease gene in the heterologous membrane but also that the transport of uracil is active and coupled to the energy furnishing system of the heterologous host.     

The role of transmembrane domain III in the lactose permease of Escherichia coli.

Deletion of putative transmembrane helix III from the lactose permease of Escherichia coli results in complete loss of transport activity. Similarly, replacement of this region en bloc with 23 contiguous Ala, Leu, or Phe residues abolishes active lactose transport. The observations suggest that helix III may contain functionally important residues; therefore, this region was subjected to Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less permease) each residue from Tyr 75 to Leu 99 was individually replaced with Cys. Twenty-one of the 25 mutants accumulate lactose to > 70% of the steady-state exhibited by C-less permease, and an additional 3 mutants transport to lower, but significant levels (40-60% of C-less). Cys replacement for Leu 76 results in low transport activity (18% of C-less). However, when placed in the wild-type background, mutant Leu 76-->Cys exhibits highly significant rates of transport (55% of wild type) and steady-state levels of lactose accumulation (65% of wild type). Immunoblots reveal that the mutants are inserted into the membrane at concentrations comparable to wild type. Studies with N-ethylmaleimide show that mutant Gly 96-->Cys is rapidly inactivated, whereas the other single-Cys mutants are not altered significantly by the alkylating agent. Moreover, the rate of inactivation of Gly 96-->Cys permease is enhanced at least 2-fold in the presence of beta-galactopyranosyl 1-thio-beta, D-galactopyranoside. The observations demonstrate that although no residue per se appears to be essential, structural properties of helix III are important for active lactose transport.     

Involvement of the central loop of the lactose permease of Escherichia coli in its allosteric regulation by the glucose-specific enzyme IIA of the phosphoenolpyruvate-dependent phosphotransferase system.

Allosteric regulation of several sugar transport systems such as those specific for lactose, maltose and melibiose in Escherichia coli (inducer exclusion) is mediated by the glucose-specific enzyme IIA (IIAGlc) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Deletion mutations in the cytoplasmic N and C termini of the lactose permease protein, LacY, and replacement of all cysteine residues in LacY with other residues did not prevent IIAGlc-mediated inhibition of lactose uptake, but several point and insertional mutations in the central cytoplasmic loop of this permease abolished transport regulation and IIAGlc binding. The results substantiate the conclusion that regulation of the lactose permease in E. coli by the PTS is mediated by a primary interaction of IIAGlc with the central cytoplasmic loop of the permease.     

Site-directed mutagenesis of tyrosine residues in the lac permease of Escherichia coli

By using oligonucleotide-directed, site-specific mutagenesis, each of the 14 Tyr residues in the lac permease of Escherichia coli was replaced with Phe, and the activity of each mutant was studied with respect to active transport, equilibrium exchange, and efflux. Ten of the mutations have no significant effect on permease activity. Of the four mutations that alter activity, replacement of Tyr26 or Tyr336 with Phe severely decreases all modes of translocation, and the binding affinity of the mutant permease for p-nitrophenyl alpha-D-galactopyranoside is markedly decreased (i.e., KD is increased). In addition, the Phe336 mutant permease is inserted into the membrane to a lesser extent than wild-type permease, as judged by immunoblot experiments. Permease containing Phe in place of Tyr236 catalyzes lactose exchange approximately 40% as well as wild-type permease but does not catalyze active transport or efflux. Finally, permease with Phe in place of Tyr382 catalyzes equilibrium exchange normally, but exhibits low rates of active transport and efflux without being uncoupled, thereby suggesting that replacement of Tyr382 with Phe alters a kinetic step involving translocation of the unloaded permease across the membrane.     

Characterization of site-directed mutants in the lac permease of Escherichia coli. 1. Replacement of histidine residues

Wild-type lac permease from Escherichia coli and two site-directed mutant permeases containing Arg in place of His35 and His39 or His322 were purified and reconstituted into proteoliposomes. H35-39R permease is indistinguishable from wild type with regard to all modes of translocation. In contrast, purified, reconstituted permease with Arg in place of His322 is defective in active transport, efflux, equilibrium exchange, and counterflow but catalyzes downhill influx of lactose without concomitant H+ translocation. Although permease with Arg in place of His205 was thought to be devoid of activity [Padan, E., Sarkar, H. K., Viitanen, P. V., Poonian, M. S., & Kaback, H. R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6765], sequencing of lac Y in pH205R reveals the presence of two additional mutations in the 5' end of the gene, and replacement of this portion of lac Y with a restriction fragment from the wild-type gene yields permease with normal activity. Permeases with Asn, Gln, or Lys in place of His322, like H322R permease, catalyze downhill influx of lactose without H+ translocation but are unable to catalyze active transport, equilibrium exchange, or counterflow. Unlike H322R permease, however, the latter mutants catalyze efflux at rates comparable to that of wild-type permease, although the reaction does not occur in symport with H+. Finally, as evidenced by flow dialysis and photoaffinity labeling experiments, replacement of His322 appears to cause a marked decrease in the affinity of the permease for substrate. The results confirm and extend the contention that His322 is the only His residue in the permease involved in lactose/H+ symport and that an imidazole moiety at position 322 is obligatory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological and regulatory properties of the general amino acid transport system of Neurospora crassa.

The fundamental properties of the general amino acid transport system of Neurospora crassa were investigated in the conidial stage of the life cycle. The transport activity was found to be under genetic control, and an isogenic set of mutants deficient for the neutral, basic, or general amino acid transport systems and combinations thereof was constructed and used for analyzing the properties specific to the general permease. Amino acid transport by this system was found to be a carrier-mediated active process with broad specificity for the neutral and basic amino acids. Kinetic analysis revealed that a common binding site functioned to transport both neutral and basic amino acids and that the permease had a high affinity for its substrates. The kinetic parameters Km, Vmax, and Ki were defined for several substrates. Two modes of regulation were detected: substrate inhibition and ammonium repression. Activity of the general system was enhanced by the removal of ammonium ions from the incubation medium with a concomitant decline in either neutral or basic permease activity, suggesting that a common component exists between the neutral and the general systems and between the basic and the general systems.