Characterization of a chromosomal mutant that blocks hemolysin excretion in Escherichia coli

Abstract Alanine dipeptides normally penetrate into Pseudomonas aeruginosa by the way of two transport systems. In peptidase N-deficient mutants, dialanine is unable to use its low affinity transport system. Uptake competition showed that this system harboured a permease common to the transport of the amino acid alanine. This permease permits the penetration of both alanine and alanyl peptides uniquely in the presence of active peptidase N. The uptake of trialanine is independent of the presence of active peptidase N inside bacteria despite the fact that hydrolysis of this tripeptide absolutely requires this activity.     

Characterization of a transport activity for long-chain peptides in barley mesophyll vacuoles

The plant vacuole is the largest compartment in a fully expanded plant cell. While only very limited metabolic activity can be observed within the vacuole, the majority of the hydrolytic activities, including proteolytic activities reside in this organelle. Since it is assumed that protein degradation by the proteasome results in the production of peptides with a size of 3-30 amino acids, we were interested to show whether the tonoplast exhibits a transport activity, which could deliver these peptides into the vacuole for final degradation. It is shown here that isolated barley mesophyll vacuoles take up peptides of 9-27 amino acids in a strictly ATP-dependent manner. Uptake is inhibited by vanadate, but not by NH(+)(4), while GTP could partially substitute for ATP. The apparent affinity for the 9 amino acid peptide was 15 μM, suggesting that peptides are efficiently transferred to the vacuole in vivo. Inhibition experiments showed that peptides with a chain length below 10 amino acids did not compete as efficiently as longer peptides for the uptake of the 9 amino acid peptide. Our results suggest that vacuoles contain at least one peptide transporter that belongs to the ABC-type transporters, which efficiently exports long-chain peptides from the cytosol into the vacuole for final degradation.     

Substrate specificity of peptide permeases in Candida albicans

Abstract Specificity of peptide transport systems in Candida albicans was studied using as an experimental tool novel anticandidal peptides, containing the N³-4-methoxyfumaroyl- l -2,3-diamino-propanoic acid residue. Studies on cross-resistance and on peptide uptake by spontaneous mutants resistant to toxic peptides, confirmed the multiplicity of peptide permeases in Candida albicans . At least two peptide permeases exist in this microorganism; the first one, specific for di- and tripeptides and the second, for oligopeptides containing 3–6 amino acids. The rate of the tritetra tetra-, penta- and hexapeptide transport in the mycelial form of Candida albicans is about 2-times higher than in the yeast form, while that of dipeptides is markedly reduced.
Tripeptides are proposed as the most efficient carriers for the delivery of 'warhead' amino acids into Candida albicans cells.     

Multiplicity of the Amino Acid Permeases in Saccharomyces cerevisiae IV. Evidence for a General Amino Acid Permease

Kinetic and genetic evidences are presented to show that, in addition to specific amino acid permeases, Saccharomyces cerevisiae has a general amino acid permease which catalyzes the transport of basic and neutral amino acids, but most probably not that of proline. The general amino acid permease appears to be constitutive, and its activity is inhibited when ammonium ions are added to the culture medium. A mutant which has lost the general amino acid permease activity was isolated. Its mutation, named gap (general amino acid permease), is not allelic to the aap (amino acid permease) mutation of Surdin et al., which has a quite different phenotype and cannot be considered as having selectively lost the general amino acid permease activity.     

Activity-dependent reversible inactivation of the general amino acid permease

The general amino acid permease, Gap1p, of Saccharomyces cerevisiae transports all naturally occurring amino acids into yeast cells for use as a nitrogen source. Previous studies have shown that a nonubiquitinateable form of the permease, Gap1p(K9R,K16R), is constitutively localized to the plasma membrane. Here, we report that amino acid transport activity of Gap1p(K9R,K16R) can be rapidly and reversibly inactivated at the plasma membrane by the presence of amino acid mixtures. Surprisingly, we also find that addition of most single amino acids is lethal to Gap1p(K9R,K16R)-expressing cells, whereas mixtures of amino acids are less toxic. This toxicity appears to be the consequence of uptake of unusually large quantities of a single amino acid. Exploiting this toxicity, we isolated gap1 alleles deficient in transport of a subset of amino acids. Using these mutations, we show that Gap1p inactivation at the plasma membrane does not depend on the presence of either extracellular or intracellular amino acids, but does require active amino acid transport by Gap1p. Together, our findings uncover a new mechanism for inhibition of permease activity in response to elevated amino acid levels and provide a physiological explanation for the stringent regulation of Gap1p activity in response to amino acids.     

Characteristics of the active transport of peptides and amino acids by germinating barley embryos

Use of two different assays involving either radioactively labelled substrates or a fluorescent-labelling procedure, gave good agreement for the rates of transport of peptides and amino acids into the scutellum of germinating grains of barley (Hordeum vulgare cv. Maris Otter, Winter). However, evidence was obtained for the enzymic decarboxylation of transpored substrate, which can cause underestimates of transport rates when using radioactively labelled substrates. The peptide Gly-Phe, was shown to be rapidly hydrolysed after uptake, and autoradiography of transported Gly-[U-¹⁴C]Phe indicated a rapid distribution of tracer, i.e. [U-¹⁴C] phenylalanine into the epithelium and sub-epithelial layers of the scutellum. The developmental patterns of transport activity indicate that peptide transport is more important nutritionally during the early stages of germination (1–3 d) whereas amino acids become relatively more important later (4–6 d). A range of amino acids is shown to be actively transported and several compete for uptake. At physiological concentrations, e.g. 2mM, transport of peptides and amino acids is inhibited about 80% by protonophore uncouplers, but at higher concentrations (10–100 mM) passive uptake predominates.Abbreviations Gly glycine - Leu leucine - Phe phenylalanine - Pro proline     

Toxicity of oxalysine and oxalysine-containing peptides against Candida albicans: regulation of peptide transport by amino acids.

A lysine antimetabolite, L-4-oxalysine [H2NCH2CH2OCH2CH(NH2)COOH], and oxalysine-containing di-, tri-, tetra- and pentapeptides inhibited growth of Candida albicans H317. Micromolar amounts of amino acids were found to overcome ammonium repression of the di- and tripeptide transport system(s) in strain H317. Several amino acids increased the toxicity of oxalysine-containing di- and tripeptides for C. albicans with little or no increase in toxicity of oxalysine or oxalysine-containing tetra- and pentapeptides. L-Lysine completely reversed the toxicity of oxalysine by competing with the transport of oxalysine into the cells. In contrast, L-lysine increased the toxicity of oxalysine-containing di- and tripeptides, but had no effect on the toxicity of oxalysine-containing tetra- and pentapeptides. Incubation of cells with L-lysine for 4 h resulted in a 15-fold increase in the rate of transport of radiolabelled dileucine, indicating that increased sensitivity of C. albicans to some toxic peptides in the presence of L-lysine may be attributed to an increased rate of transport of these peptides. Our results indicate that the dipeptide and tripeptide transport system(s) of C. albicans are regulated by micromolar amounts of amino acids in a similar fashion to the regulation of peptide transport in Saccharomyces cerevisiae and that multiple peptide transport systems differentially regulated by various nitrogen sources and amino acids exist in C. albicans.     

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.     

Compensating stereochemical changes allow murein tripeptide to be accommodated in a conventional peptide-binding protein

The oligopeptide permease (Opp) of Escherichia coli is an ATP-binding cassette transporter that uses the substrate-binding protein (SBP) OppA to bind peptides and deliver them to the membrane components (OppBCDF) for transport. OppA binds conventional peptides 2-5 residues in length regardless of their sequence, but does not facilitate transport of the cell wall component murein tripeptide (Mtp, L-Ala-γ-D-Glu-meso-Dap), which contains a D-amino acid and a γ-peptide linkage. Instead, MppA, a homologous substrate-binding protein, forms a functional transporter with OppBCDF for uptake of this unusual tripeptide. Here we have purified MppA and demonstrated biochemically that it binds Mtp with high affinity (K(D) ～ 250 nM). The crystal structure of MppA in complex with Mtp has revealed that Mtp is bound in a relatively extended conformation with its three carboxylates projecting from one side of the molecule and its two amino groups projecting from the opposite face. Specificity for Mtp is conferred by charge-charge and dipole-charge interactions with ionic and polar residues of MppA. Comparison of the structure of MppA-Mtp with structures of conventional tripeptides bound to OppA, reveals that the peptide ligands superimpose remarkably closely given the profound differences in their structures. Strikingly, the effect of the D-stereochemistry, which projects the side chain of the D-Glu residue at position 2 in the direction of the main chain in a conventional tripeptide, is compensated by the formation of a γ-linkage to the amino group of diaminopimelic acid, mimicking the peptide bond between residues 2 and 3 of a conventional tripeptide.     

PTR3, a novel gene mediating amino acid-inducible regulation of peptide transport in Saccharomyces cerevisiae

We have isolated and characterized the Saccharomyces cerevisiae PTR3 gene by functional complementation of a mutant deficient for amino acid-inducible peptide transport. PTR3 is predicted to encode a protein of 678 amino acids that exhibits no similarity to any other protein in the database. Deletion of the PTR3 open reading frame pleiotropically reduced the sensitivity to toxic peptides and amino acid analogues. Initial rates of radiolabelled dipeptide uptake demonstrated that elimination of PTR3 resulted in the loss of amino acid-induced levels of peptide transport. PTR3 was required for amino acid-induced expression of PTR2 , the gene encoding the dipeptide/tripeptide transport protein, but was not necessary for nitrogen catabolite repression of peptide import or PTR2 expression. It was determined that PTR3 also modulates expression of BAP2 , the gene encoding the branched-amino acid permease. Furthermore, we present genetic evidence that suggests that PTR3 functions within a novel regulatory pathway that facilitates amino acid induction of the PTR system.     

Modular structure of genes encoding multifunctional peptide synthetases required for non-ribosomal peptide synthesis

Abstract Peptide synthetases are large multienzyme complexes that catalyze the non-ribosomal synthesis of a structurally diverse family of bioactive peptides. They possess a multidomain structure and employ the thiotemplate mechanism to activate, modify and link together by amide or ester bonds the constituent amino acids of the peptide product. The domains, which represent the functional building units of peptide synthetases, appear to act as independent enzymes whose specific linkage order forms the protein-template that defines the sequence of the incorporated amino acids. Two types of domains have been characterized in peptide synthetases of bacterial and fungal origin: type I comprises about 600 amino acids and contains at least two modules involved in substrate recognition, adenylation and thioester formation, whereas type II domains carry in addition an insertion of about 430 amino acids that may function as a N-methyltransferase module. The role of other genes associated with bacterial opérons encoding peptide synthetases is also discussed.     

Random mutagenesis of the prokaryotic peptide transporter YdgR identifies potential periplasmic gating residues

The peptide transporter (PTR) family represents a group of proton-coupled secondary transporters responsible for bulk uptake of amino acids in the form of di- and tripeptides, an essential process employed across species ranging from bacteria to humans. To identify amino acids critical for peptide transport in a prokaryotic PTR member, we have screened a library of mutants of the Escherichia coli peptide transporter YdgR using a high-throughput substrate uptake assay. We have identified 35 single point mutations that result in a full or partial loss of transport activity. Additional analysis, including homology modeling based on the crystal structure of the Shewanella oneidensis peptide transporter PepT(so), identifies Glu(56) and Arg(305) as potential periplasmic gating residues. In addition to providing new insights into transport by members of the PTR family, these mutants provide valuable tools for further study of the mechanism of peptide transport.     

Structural requirements for microvascular permeability-increasing ability of peptides: Studies on analogues of a fibrinogen pentapeptide fragment

A pentapeptide, Ala-Arg-Pro-Ala-Lys, liberated from fibrinogen during plasmin-mediated fibrinolysis, was shown earlier to increase microvascular permeability in rat and human skin. Eighteen new analogues have now been synthesized in addition to the 15 previously prepared and examined for their effect on permeability. The old concept that a tetrapeptide with basic amino acids at both ends and a proline residue adjacent to the N-terminal amino acid is essential for high activity on permeability, has now been challenged. The results obtained with several of the new analogues strengthen this concept. More interestingly, however, the third amino acid, which was found in earlier studies to be less sensitive to exchange, has now been deleted as well as duplicated with only a modest loss of activity of the peptide. The chirality of the C-terminal amino acid, most surprisingly, does not seem to be crucial for peptide activity. Slightly superpotent analogues were obtained on amidation of the C-terminus. In addition, a few naturally occurring peptides, namely tuftsin, substance P, neurotensin and bradykinin, the amino acid sequences of which all exhibit characteristic features of some of our active peptide analogues were investigated in the same test system. Tuftsin displayed a potency equal to that of the pentapeptide. The other three peptides were all highly superpotent in this assay system.     

A leucine zipper-based peptide hybrid delivers functional Nanog protein inside the cell nucleus

We synthesized a pair of compounds containing leucine zipper peptides to deliver protein cargo into cells. One is a cell-penetrating peptide (CPP) with Lz(E), a leucine zipper peptide containing negatively charged amino acids, and the other is a Nanog protein with Lz(K), a leucine zipper peptide containing positively charged amino acids. When cells were treated with these equimolar mixtures, Nanog-Lz(K) hybridized with Lz(E)-CPP was successfully delivered into the cells. Furthermore, Nanog-Lz(K) exerted its proper function after nuclear transport.     

Multiplicity of oligopeptide transport systems in Escherichia coli.

The ability of Escherichia coli K-12 4212 to utilize a variety of oligopeptides as sources of required amino acids was examined. Triornithine-resistant mutants of this strain were oligopeptide permease deficient (Opp-) as judged by their inability to utilize (Lys)3 and (Lys)4 as sources of lysine and their resistance to the toxic tripeptide (Val)3. These same mutants were able to grow when Met-Met-Met, Met-Gly-Met, Met-Gly-Gly, Gly-Met-Gly, Gly-Gly-Met, Gly-Met-Met, Met-Met-Gly, or Leu-Leu-Leu were supplied in place of the requisite amino acid. The system mediating the uptake of these peptides, herein designated Opr I, was not able to transport N-alpha-acetylated peptides, nor the tetrapeptides Met-Gly-Met-Met, Met-Met-Gly-Met, or Met-Met-Met-Gly. Competition experiments indicated that trimethionine and trileucine enter E. coli K-12 via either Opp or Opr I. Analogous results were found using the methionine, leucine-requiring auxotroph E. coli B163. It appears that more than one oligopeptide transport system exists in E. coli and that the system mediating peptide uptake is complex.     

Modifications of substituted seryl and threonyl residues in phosphopeptides and a polysialoglycoprotein by beta-elimination and nucleophile additions

The beta-elimination and nucleophile addition reactions of the substituted serine and threonine residues were studied using several synthesized fluorescence-labeled phosphopeptides and a salmon egg polysialoglycoprotein (PSGP). The reagents used were 1 M CH3SH-0.43 M NaOH, 1 M NaBH4-0.1 M NaOH, 1 M CH3NH2-0.1 M NaOH, and 1 M Na2SO3-0.1 M NaOH. The beta-elimination reaction of a phosphoserine peptide, Gly-Ser(PO4)-Glu-AEAP, was about 20 times faster than that of the corresponding phosphothreonine peptide. The carboxyl-side amino acid of the phosphoamino acids in peptides greatly affected the beta-elimination rate. The beta-elimination reaction rates of O-glycosyl serine and threonine in the polysialoglycoprotein were similar and were about a half of that of the phosphoserine peptide. The rates of addition of the three nucleophiles and hydrogen to alpha-aminoacrylic acid (beta-elimination product of substituted serine) in the peptide decreased in the order of CH3SH, Na2SO3, CH3NH2, and H2(NaBH4), and the addition to alpha-aminocrotonic acid (beta-elimination product of substituted threonine) in the order of Na2SO3, CH3NH2, CH3SH, and H2. These results indicated that sulfite is the most recommended nucleophile because of its high addition rate. If sulfite addition is carried out in the presence of NaBH4, sugar chains can be released as alditols, converting the sugar-attaching amino acids to beta-sulfoamino acids.     

Uptake of cell wall peptides by Salmonella typhimurium and Escherichia coli.

During bacterial growth, cell wall peptides are released from the murein and reused for the synthesis of new cell wall material. Mutants defective in peptide transport were unable to reutilize cell wall peptides, demonstrating that these peptides are taken up intact into the cytoplasm prior to reincorporation into murein. Furthermore, cell wall peptide recycling was shown to play an important physiological role; peptide transport mutants which were unable to recycle these peptides showed growth defects under appropriate conditions. Using mutants specifically defective in each of the three peptide transport systems, we showed that the uptake of cell wall peptides was mediated solely by the oligopeptide permease (Opp) and that neither the dipeptide permease (Dpp) nor the tripeptide permease (Tpp) played a significant role in this process. Our data indicate that the periplasmic oligopeptide-binding protein has more than one substrate-binding site, each with different though overlapping specificities.     

Peptide uptake in germinating barley embryos involves a dithiol-dependent transport protein

An active transport system for small peptides occurs in the scutellar membrane of germinating barley and serves to move the products of partial hydrolysis of storage proteins from the endosperm into the growing embryo. Transport of peptides, but not amino acids or glucose, is inhibited by the thiol reagents, N-ethylmaleimide and p-chloromercuribenzene sulphonic acid (PCMBS). Peptide substrates protect against PCMBS inactivation. The dithiol-specific reagent, phenylarsine oxide (PAO) also inhibits. The reducing agent, dithiothreitol, reverses the inactivation caused by PCMBS and PAO. We conclude that the peptide transport system contains a redox-sensitive, dithiol-dependent protein.     

Characterization of AAT1: a gene involved in the regulation of amino acid transport in Saccharomyces cerevisiae

A new class of Saccharomyces cerevisiae mutants (aat1 - amino acid transport) has been identified. These mutants are unable to grow on rich medium or on minimal medium supplemented with certain amino acids (isoleucine, methionine, phenylalanine, tyrosine or valine). This phenotype is directly linked to the presence of the leu2 allele in these strains: aat1 LEU2 organisms grow normally on all media tested. Leucine uptake through the leucine-specific permease is inhibited to less than 35% of wild-type levels in aat1 cells preincubated in nonpermissive media, and the activity of the general amino acid permease is also low in these conditions. aat1 cells are therefore unable to grow on rich media because they cannot take up enough leucine to supplement their auxotrophic requirement.     

Stereospecificity of peptide transport by germinating barley embryos

The stereospecific requirements for peptide transport in the scutellum of germinating barley (Hordeum vulgare) embryos are described. Replacement of an L-amino acid residue in a peptide by its D-stereoisomer decreases the affinity of the peptide for the transport site, leading to a reduction in transport. Substitution of a second D-residue reduces affinity still further. The extent to which transport is inhibited depends upon the position of the D-residue in the primary sequence, with D-residues at the C-terminus of the peptide having the greatest effect. Competition between D- and L-peptides indicates that they both enter via the same transport system. Although D-amino acids can be accumulated when presented as a peptide, these same D-residues are not transported when supplied as the free amino acids. L-Leu-D-leu is accumulated intact against a concentration gradient, indicating the operation of an active transport mechanism that can function without the involvement of peptidase activity.