Phaseolotoxin transport in Escherichia coli and Salmonella typhimurium via the oligopeptide permease.

Phaseolotoxin [(N delta-phosphosulfamyl)ornithylalanylhomoarginine], a phytotoxic tripeptide produced by Pseudomonas syringae pv. phaseolicola that inhibits ornithine carbamoyltransferase, is transported into Escherichia coli and Salmonella typhimurium via the oligopeptide transport system (Opp). Mutants defective in oligopeptide permease (Opp-) were resistant to phaseolotoxin. Spontaneous phaseolotoxin-resistant mutants (Toxr) lacked the Opp function as evidenced by their cross-resistance to triornithine and failure to utilize glycylhistidylglycine as a source of histidine. Growth inhibition by phaseolotoxin was prevented by peptides known to be transported via the Opp system and by treatment of the toxin with L-aminopeptidase. In both E. coli and S. typhimurium, Toxr mutations were cotransducible with trp, suggesting that the opp locus occupies similar positions in genetic maps of these bacteria.     

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.     

Molecular characterization of the oligopeptide permease of Salmonella typhimurium

The oligopeptide permease (Opp) of Salmonella typhimurium is a periplasmic binding protein-dependent transport system and handles any peptides containing from two to five amino acid residues. Opp plays an important nutritional role and is also required for the recycling of cell wall peptides. We have determined the nucleotide sequence of the opp operon. In addition to the four opp genes identified previously by genetic means (oppABCD) a fifth gene, oppF, is shown to be cotranscribed as part of the opp operon. Using reverse genetics, we show that oppF also encodes an essential component of the Opp transport system. The five proteins, OppABCDF, are shown to be the only proteins required for Opp function. Regulation of opp expression and of the differential expression of genes within the operon is investigated. We have devised a simple means of constructing lacZ gene fusions to any S. typhimurium chromosomal gene in vivo, using derivatives of bacteriophage Mu. Using this procedure, opp-lacZ gene fusions were selected. The resultant Opp-LacZ hybrid proteins were used to show that OppB, OppC and OppD are membrane-associated proteins. A detailed comparison of the Opp components with those of other binding protein-dependent transport systems provides insight into the mechanisms and evolution of these transport systems.     

Specificity of peptide transport systems in Lactococcus lactis: evidence for a third system which transports hydrophobic di- and tripeptides.

A proton motive force-driven di-tripeptide carrier protein (DtpT) and an ATP-dependent oligopeptide transport system (Opp) have been described for Lactococcus lactis MG1363. Using genetically well-defined mutants in which dtpT and/or opp were inactivated, we have now established the presence of a third peptide transport system (DtpP) in L. lactis. The specificity of DtpP partially overlaps that of DtpT. DtpP transports preferentially di- and tripeptides that are composed of hydrophobic (branched-chain amino acid) residues, whereas DtpT has a higher specificity for more-hydrophilic and charged peptides. The toxic dipeptide L-phenylalanyl-beta-chloro-L-alanine has been used to select for a di-tripeptide transport-negative mutant with the delta dtpT strain as a genetic background. This mutant is unable to transport di- and tripeptides but still shows uptake of amino acids and oligopeptides. The DtpP system is induced in the presence of di- and tripeptides containing branched-chain amino acids. The use of ionophores and metabolic inhibitors suggests that, similar to Opp, DtpP-mediated peptide transport is driven by ATP or a related energy-rich phosphorylated intermediate.     

Functional testing of putative oligopeptide permease (Opp) proteins of Borrelia burgdorferi: a complementation model in opp(-) Escherichia coli

Studies of the protein function of Borrelia burgdorferi have been limited by a lack of tools for manipulating borrelial DNA. We devised a system to study the function of a B. burgdorferi oligopeptide permease (Opp) orthologue by complementation with Escherichia coli Opp proteins. The Opp system of E. coli has been extensively studied and has well defined substrate specificities. The system is of interest in B. burgdorferi because analysis of its genome has revealed little identifiable machinery for synthesis or transport of amino acids and only a single intact peptide transporter operon. As such, peptide uptake may play a major role in nutrition for the organism. Substrate specificity for ABC peptide transporters in other organisms is determined by their substrate binding protein. The B. burgdorferi Opp operon differs from the E. coli Opp operon in that it has three separate substrate binding proteins, OppA-1, -2 and -3. In addition, B. burgdorferi has two OppA orthologues, OppA-4 and -5, encoded on separate plasmids. The substrate binding proteins interact with integral membrane proteins, OppB and OppC, to transport peptides into the cell. The process is driven by two ATP binding proteins, OppD and OppF. Using opp-deleted E. coli mutants, we transformed cells with B. burgdorferi oppA-1, -2, -4 or -5 and E. coli oppBCDF. All of the B. burgdorferi OppA proteins are able to complement E. coli OppBCDF to form a functional Opp transport system capable of transporting peptides for nutritional use. Although there is overlap in substrate specificities, the substrate specificities for B. burgdorferi OppAs are not identical to that of E. coli OppA. Transport of toxic peptides by B. burgdorferi grown in nutrient-rich medium parallels borrelial OppA substrate specificity in the complementation system. Use of this complementation system will pave the way for more detailed studies of B. burgdorferi peptide transport than currently available tools for manipulating borrelial DNA will allow.     

Identification of a second oligopeptide transport system in Bacillus subtilis and determination of its role in sporulation

Summary
Sporulation in Bacillus subtilis depends on an intact oligopeptide transport system, the Opp system. Mutants in opp sporulate poorly but second-site revertants can be found that restore sporulation and pep-tide transport. These second-site mutations were found in a second oligopeptide transport system, app , in which the peptide-binding protein, AppA, is mutant owing to a frame-shift mutation, and the revertants restore the original frame. The AppA mutation is present in the 168 strain of B. subtilis. The app operon consists of five genes in the order appD-appF-appA-appB-appC , with the locus designations corresponding to their homologue in the opp operon. Homology between the app and opp proteins ranges from 54% identity for AppF and OppF, to 22% identity for AppA and OppA. Both the App and Opp permease systems can transport tetra- and pentapeptides, but tripeptides are not transported by the App system. Strains of the genotype app ⁺opp⁻ are resistant to the tripeptide antibiotic bialaphos. The repaired App system can substitute completely for the Opp system in both sporulation and competence for genetic transformation. The pheno-types raised some speculation about the subunit configuration of the Opp system.     

Specialized peptide transport system in Escherichia coli.

Trileucine is utilized as a source of leucine for growth of strains of Escherichia coli K-12 that are deficient in the oligopeptide transport system (Opp). Trithreonine is toxic to E. coli K-12. Opp- mutants of E. coli K-12 retain complete sensitivity to this tripeptide. Moreover, E. coli W, which is resistant to trithreonine, can utlize this tripeptide as a threonine source and this capability is fully maintained in E. coli W (Opp-). A spontaneous trithreonine-resistant mutant of E. coli K-12 (Opp-) has been isolated that has an impaired growth response to trileucine and is resistant to trithreonine. Trileucine competes with the uptake of trithreonine as measured by its ability to relieve trithreonine toxicity in E. coli K-12. It is concluded that trileucine as well as trithreonine are transported into E. coli K-12 or W by a common uptake system that is distinct from the Opp system. Trimethionine can act as a competitor of trileucine or trithreonine-supported growth and as an antagonist of trithreonine toxicity in Opp- mutants. It is concluded that trimethionine is recognized by the trileucine-trithreonine transport system. Trithreonine, trimethionine, and trileucine are also transported by the Opp system, as they all relieve triornithine toxicity towards E. coli W and compete with tetralysine utilization as lysine source for growth of a lysine auxotroph of this strain.     

Escherichia coli K-12 Mutants Altered in the Transport Systems for Oligo- and Dipeptides

Two mutants of Escherichia coli K-12, defective in the oligopeptide and dipeptide transport system, are described. A mutant defective in the oligopeptide transport system (opp-1) was isolated as resistant to the inhibitory action of triornithine; this mutant is also resistant to glycylglycylvaline and does not concentrate (14)C-glycylglycylglycine, although it is still as sensitive as the parental strain to glycylvaline and valine. Starting from the opp-1 strain, a mutant defective also in the dipeptide transport system (dpp-1) was isolated; this mutant is resistant to the inhibitory action of glycylvaline, valylleucine, and leucylvaline and does not concentrate (14)C-glycylglycine, although it is still as sensitive as the parental strain to valine. The apparent kinetic constants for oligopeptide and dipeptide transport were measured. The opp marker is co-transducible with trp at 27 min on the E. coli genetic map. The dpp locus is separated from opp and is located between proC (10 min) and opp.     

Oligopeptide uptake and aminoglycoside resistance in Escherichia coli K12

Previous reports have suggested that Escherichia coli K12 mutants defective in the expression of oligogopeptide permease protein A (OppA) exhibit reduced sensitivity to aminoglycosides due to altered permeability of the cell envelope. In this work, the role of the OppA protein, and the oligogopeptide permease (Opp) transport system has been evaluated, in the resistance to aminoglycosides using derivatives of the E. coli K12 SS320 strain selected for triornithine resistance or with a deletion of the complete opp operon. All tested mutants were defective in the uptake of tri- and tetra-peptides but did not expressed resistance to aminoglycosides. Additionally, complementation tests carried out with a plasmid encoding the OppA protein did not affect the sensitivity of the strains to these antibiotics. Taken together, these evidences indicate that the Opp uptake system, as well as the OppA protein, does not play a direct role in the sensitivity to aminoglycosides in E. coli K12.     

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.     

Genetic analysis of Escherichia coli oligopeptide transport mutants.

The composition of the outer membrane channels formed by the OmpF and OmpC porins is important in peptide permeation, and elimination of these proteins from the Escherichia coli outer membrane results in a cell in which the primary means for peptide permeation through this cell structure has been lost. E. coli peptide transport mutants which harbor defects in genes other than the ompF/ompC genes have been isolated on the basis of their resistance to toxic tripeptides. The genetic defects carried by these oligopeptide permease-negative (Opp-) strains were found to map in two distinct chromosomal locations. One opp locus was trp linked and mapped to the interval between att phi 80 and galU. Complementation studies with F'123 opp derivatives indicated that this peptide transport locus resembles that characterized in Salmonella typhimurium as a tetracistronic operon (B. G. Hogarth and C. F. Higgins, J. Bacteriol. 153:1548-1551, 1983). The second opp locus, which we have designated oppE, was mapped to the interval between dnaC and hsd at 98.5 min on the E. coli chromosome. The differences in peptide utilization, sensitivity and resistance to toxic peptides, and the L-[U-14C]alanyl-L-alanyl-L-alanine transport properties observed with these Opp-E. coli strains demonstrated that the transport systems encoded by the trp-linked opp genes and by the oppE gene(s) have different substrate preferences. Mutants harboring defects in both peptide transport loci defined in this study would not grow on nutritional peptides except for tri-L-methionine, were totally resistant to toxic peptides, and would not actively transport L-[U-14C]alanyl-L-alanyl-L-alanine.     

Functional testing of putative oligopeptide permease (Opp) proteins of Borrelia burgdorferi: a complementation model in opp− Escherichia coli

Studies of the protein function of Borrelia burgdorferi have been limited by a lack of tools for manipulating borrelial DNA. We devised a system to study the function of a B. burgdorferi oligopeptide permease (Opp) orthologue by complementation with Escherichia coli Opp proteins. The Opp system of E. coli has been extensively studied and has well defined substrate specificities. The system is of interest in B. burgdorferi because analysis of its genome has revealed little identifiable machinery for synthesis or transport of amino acids and only a single intact peptide transporter operon. As such, peptide uptake may play a major role in nutrition for the organism. Substrate specificity for ABC peptide transporters in other organisms is determined by their substrate binding protein. The B. burgdorferi Opp operon differs from the E. coli Opp operon in that it has three separate substrate binding proteins, OppA-1, -2 and -3. In addition, B. burgdorferi has two OppA orthologues, OppA-4 and -5, encoded on separate plasmids. The substrate binding proteins interact with integral membrane proteins, OppB and OppC, to transport peptides into the cell. The process is driven by two ATP binding proteins, OppD and OppF. Using opp-deleted E. coli mutants, we transformed cells with B. burgdorferi oppA-1, -2, -4 or -5 and E. coli oppBCDF. All of the B. burgdorferi OppA proteins are able to complement E. coli OppBCDF to form a functional Opp transport system capable of transporting peptides for nutritional use. Although there is overlap in substrate specificities, the substrate specificities for B. burgdorferi OppAs are not identical to that of E. coli OppA. Transport of toxic peptides by B. burgdorferi grown in nutrient-rich medium parallels borrelial OppA substrate specificity in the complementation system. Use of this complementation system will pave the way for more detailed studies of B. burgdorferi peptide transport than currently available tools for manipulating borrelial DNA will allow.     

Peptide substrates rapidly modulate expression of dipeptide and oligopeptide permeases in Candida albicans

Previous studies showed that peptide transport activity in Candida albicans was completely repressed by NH4+, and that growth on amino acids as sole nitrogen source stimulated transport to a basal level. Here we show that addition of peptide mixtures to culture media gives a further 5-fold increase in transport of dipeptides and oligopeptides; the effect is specific for peptide transport, amino acid uptake being unaffected. Presence of peptides but not amino acids overrides NH4+ repression of peptide transport. Step-up activation of transport activity, caused by addition of peptides to incubation media, and step-down inhibition that accompanies removal of peptides, occurs rapidly (within 30 min at 28 degrees C). Step-up is independent of de novo protein synthesis. This substrate-induced regulation is compatible with a rapid, reversible activation of plasma membrane-bound peptide permease(s), or a mechanism of endocytosis involving a cycle of insertion and retrieval of preformed permease components. These results are considered in relation to the expression of peptide permeases in vivo, and the development of synthetic anticandidal peptide carrier prodrugs designed to exploit these systems.     

Reconstruction of the proteolytic pathway for use of β-casein by Lactococcus lactis

Amino acid auxotrophous bacteria such as Lactococcus lactis use proteins as a source of amino acids. For this process, they possess a complex proteolytic system to degrade the protein(s) and to transport the degradation products into the cell. We have been able to dissect the various steps of the pathway by deleting one or more genes encoding key enzymes/components of the system and using mass spectrometry to analyse the complex peptide mixtures. This approach revealed in detail how L . lactis liberates the required amino acids from β-casein, the major component of the lactococcal diet. Mutants containing the extracellular proteinase PrtP, but lacking the oligopeptide transport system Opp and the autolysin AcmA, were used to determine the proteinase specificity in vivo . To identify the substrates of Opp present in the casein hydrolysate, the PrtP-generated peptide pool was offered to mutants lacking the proteinase, but containing Opp, and the disappearance of peptides from the medium as well as the intracellular accumulation of amino acids and peptides was monitored in peptidase-proficient and fivefold peptidase-deficient genetic backgrounds. The results are unambiguous and firmly establish that (i) the carboxyl-terminal end of β-casein is degraded preferentially despite the broad specificity of the proteinase; (ii) peptides smaller than five residues are not formed in vivo  ; (iii) use of oligopeptides of 5–10 residues becomes only possible after uptake via Opp; (iv) only a few (10–14) of the peptides generated by PrtP are actually used, even though the system facilitates the transport of oligopeptides up to at least 10 residues. The technology described here allows us to monitor the fate of individual peptides in complex mixtures and is applicable to other proteolytic systems.     

Charged casein-derived oligopeptides competitively inhibit the transport of a reporter oligopeptide by Lactococcus lactis

AIM: To study the effect of casein-derived peptides, accumulated during growth of Lactococcus lactis in milk, on its oligopeptide transport (Opp) function. METHODS AND RESULTS: This effect was estimated by analysing the ability of casein-derived peptides to compete for the transport of a reporter peptide by whole L. lactis cells. The transport of the reported peptide was monitored by determining the intracellular concentrations of the corresponding amino acids by means of reverse-phase high-performance liquid chromatography (HPLC). Uptake of the reporter peptide was competitively inhibited by casein-derived peptides. The competition was only because of charged casein-derived peptides, including anionic peptides. The design of specific pure peptides made it possible to evidence for a positive (or negative) influence exerted by the positively (or negatively) charged side chain of the N-terminal amino acid on the competition. CONCLUSIONS: Charged casein-derived peptides impaired the oligopeptide transport function of L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate an inhibition of Opp when too many peptides are produced by the proteinase. Peptide transport by Opp therefore represents a bottleneck for increasing the growth rate of L. lactis in milk.