FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1978360923> ?p ?o ?g. }

• W1978360923 endingPage "14840" @default.
• W1978360923 startingPage "14832" @default.
• W1978360923 abstract "The specific oligopeptide transport system Opp is essential for growth of Lactococcus lactis in milk. We examined the biodiversity of oligopeptide transport specificity in theL. lactis species. Six strains were tested for (i) consumption of peptides during growth in a chemically defined medium and (ii) their ability to transport these peptides. Each strain demonstrated some specific preferences for peptide utilization, which matched the specificity of peptide transport. Sequencing of the binding protein OppA in some strains revealed minor differences at the amino acid level. The differences in specificity were used as a tool to unravel the role of the binding protein in transport specificity. The genes encoding OppA in four strains were cloned and expressed inL. lactis MG1363 deleted for its oppA gene. The substrate specificity of these engineered strains was found to be similar to that of the L. lactis MG1363 parental strain, whichever oppA gene was expressed. In situbinding experiments demonstrated the ability of OppA to interact with non-transported peptides. Taken together, these results provide evidence for a new concept. Despite that fact that OppA is essential for peptide transport, it is not the (main) determinant of peptide transport specificity in L. lactis. The specific oligopeptide transport system Opp is essential for growth of Lactococcus lactis in milk. We examined the biodiversity of oligopeptide transport specificity in theL. lactis species. Six strains were tested for (i) consumption of peptides during growth in a chemically defined medium and (ii) their ability to transport these peptides. Each strain demonstrated some specific preferences for peptide utilization, which matched the specificity of peptide transport. Sequencing of the binding protein OppA in some strains revealed minor differences at the amino acid level. The differences in specificity were used as a tool to unravel the role of the binding protein in transport specificity. The genes encoding OppA in four strains were cloned and expressed inL. lactis MG1363 deleted for its oppA gene. The substrate specificity of these engineered strains was found to be similar to that of the L. lactis MG1363 parental strain, whichever oppA gene was expressed. In situbinding experiments demonstrated the ability of OppA to interact with non-transported peptides. Taken together, these results provide evidence for a new concept. Despite that fact that OppA is essential for peptide transport, it is not the (main) determinant of peptide transport specificity in L. lactis. L. lactis OppA chemically defined medium high pressure liquid chromatography The oligopeptide transport system Opp has been described in many bacteria. This transport system may be involved in (i) nutrient acquisition in Lactococcus lactis (1Juillard V. Le Bars D. Kunji E.R.S. Konings W.N. Gripon J.-C. Richard J. Appl. Environ. Microbiol. 1995; 61: 3024-3030Crossref PubMed Google Scholar, 2Kunji E.R.S. Hagting A. De Vries C.J. Juillard V. Haandrikman A.J. Poolman B. Konings W.N. J. Biol. Chem. 1995; 270: 1569-1574Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) orStreptococcus thermophilus (3Garault P. Le Bars D. Besset C. Monnet V. J. Biol. Chem. 2002; 277: 32-39Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar); (ii) recycling of cell wall peptides for peptidoglycan synthesis in Salmonella typhimurium and Escherichia coli (4Goodell E.W. Higgins C.F. J. Bacteriol. 1987; 169: 3861-3865Crossref PubMed Google Scholar); (iii) sensing of extracellular signaling molecules (pheromones) required for the initiation of competence and sporulation in Bacillus subtilis (5Perego M. Higgins C.F. Pearce S.R. Gallagher M.P. Hoch A.J. Mol. Microbiol. 1991; 5: 173-185Crossref PubMed Scopus (240) Google Scholar, 6Koı̈de A. Koch J.A. Mol. Microbiol. 1994; 13: 417-426Crossref PubMed Scopus (83) Google Scholar, 7Solomon J.M. Magnusson R. Srivastava A. Grossman A.D. Genes Dev. 1995; 9: 547-558Crossref PubMed Scopus (158) Google Scholar, 8Lazazzera B.A. Kurster I.G. McQuade R.S. Grossman A.D. J. Bacteriol. 1999; 181: 5193-5200Crossref PubMed Google Scholar), for the induction of conjugation inEnterococcus faecalis (9Bensing B.A. Manias D.A. Dunny G.M. Mol. Microbiol. 1997; 24: 285-294Crossref PubMed Scopus (39) Google Scholar, 10Leonard B.A. Podbielski P. Hedberg P.J. Dunny G.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 260-264Crossref PubMed Scopus (157) Google Scholar), and for the induction of virulence in several pathogenic bacteria (11Podbielski A. Pohl B. Woischnick M. Körner C. Schmidt K.H. Rozdzinski E. Leonard B.A.B. Mol. Microbiol. 1996; 21: 1087-1099Crossref PubMed Scopus (87) Google Scholar, 12Coulter S.N. Schwan W.R. Ng E.Y. Langhorne M.H. Ritchie H.D. Westbrock-Wadman S. Hufnagle W.O. Folger K.R. Bayer A.S. Stover C.K. Mol. Microbiol. 1998; 30: 393-404Crossref PubMed Scopus (236) Google Scholar, 13Claverys J.P. Grossiord B. Alloing G. Res. Microbiol. 2000; 151: 457-463Crossref PubMed Scopus (44) Google Scholar, 14Gominet M. Slamti L. Gilois N. Rose M. Lereclus D. Mol. Microbiol. 2001; 40: 963-975Crossref PubMed Scopus (144) Google Scholar); and (iv) growth at low temperatures and intracellular survival in macrophages ofListeria monocytogenes (15Borezee E. Pellegrini E. Berche P. Infect. Immun. 2000; 68: 7069-7077Crossref PubMed Scopus (161) Google Scholar). Opp is a member of a superfamily of highly conserved ATP-binding cassette transporters. In Gram-negative bacteria, the transporter comprises a periplasmic solute-binding protein (OppA) and a translocon consisting of two integral membrane proteins (OppB and OppC) and two membrane-bound cytoplasmic ATP-binding proteins (OppD and OppF). In Gram-positive bacteria, OppA proteins are lipoproteins anchored to the cell membrane by their N-terminal lipid moiety. Although several copies of the gene encoding the binding protein might be present in Gram-positive bacteria (3Garault P. Le Bars D. Besset C. Monnet V. J. Biol. Chem. 2002; 277: 32-39Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 16Bolotine A. Wincker P. Mauger S. Jaillon O. Malarme K. Weissenbach J. Ehrlich S.D. Sorokin A. Genome Res. 2001; 11: 731-753Crossref PubMed Scopus (964) Google Scholar), only one seems to be functional inL. lactis (17Tynkkynen S. Buist G. Kunji E. Kok J. Poolman B. Venema G. Haandrikman A. J. Bacteriol. 1993; 175: 7523-7532Crossref PubMed Google Scholar, 18Yu W. Gillies K. Kondo J.K. Broadbent J.R. Mc Kay L.L. Plasmid. 1996; 35: 145-155Crossref PubMed Scopus (29) Google Scholar). OppA serves as an initial receptor. It binds the substrate and delivers it to the transmembrane complex. It is generally considered as the specificity determinant of the system, whereas the rate of peptide transport is imposed by the rate of peptide donation from OppA to the OppBC complex (19Lanfermeijer F.C. Picon A. Konings W.N. Poolman B. Biochemistry. 1999; 38: 14440-14450Crossref PubMed Scopus (47) Google Scholar). The ATP-binding proteins couple ATP hydrolysis to the transport process. The substrate specificity of Opp from S. typhimurium has been well established. S. typhimurium Opp transports peptides from two to five amino acids with a broad range of sequences (20Tame J.R.H. Dodson E.J. Murshudov G. Higgins C.F. Wilkinson A.J. Structure. 1995; 3: 1395-1406Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). S. typhimurium OppA has a higher affinity for tripeptides than for dipeptides (21Rostom A.A. Tame J.R.H. Ladbury J.E. Robinson C.V. J. Mol. Biol. 2000; 296: 269-279Crossref PubMed Scopus (48) Google Scholar). The Gram-positive bacteriumL. lactis shows significant differences in peptide uptake and affinity compared with S. typhimurium. First of all,L. lactis MG1363 is able to transport oligopeptides containing up to 18 amino acids (22Detmers F.J.M. Kunji E.R.S. Lanfermeijer F.C. Poolman B. Konings W.N. Biochemistry. 1998; 37: 16671-16679Crossref PubMed Scopus (76) Google Scholar). Its binding protein preferentially interacts with nonameric peptides, but is able to bind peptides containing up to 35 amino acid residues (23Detmers F.J.M. Lanfermeijer F.C. Abele R. Jack R.W. Tampé R. Konings W.N. Poolman B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12487-12492Crossref PubMed Scopus (63) Google Scholar). Nevertheless, the strain preferentially uses hydrophobic basic peptides with molecular masses ranging between 600 and 1100 Da (24Juillard V. Guillot A. Le Bars D. Gripon J.-C. Appl. Environ. Microbiol. 1998; 64: 1230-1236Crossref PubMed Google Scholar), whereas di- and tripeptides are not transported by the L. lactis Opp system (16Bolotine A. Wincker P. Mauger S. Jaillon O. Malarme K. Weissenbach J. Ehrlich S.D. Sorokin A. Genome Res. 2001; 11: 731-753Crossref PubMed Scopus (964) Google Scholar, 25Kunji E.R.S. Smid E.J. Plapp R. Poolman B. Konings W.N. J. Bacteriol. 1993; 175: 2052-2059Crossref PubMed Google Scholar). In this work, we compared the ability of different strains of L. lactis to transport oligopeptides. In the L. lactisspecies, we demonstrate the existence of variability in both the specificity of peptide transport and the amino acid sequence ofL. lactis OppA (OppALl).1 In an attempt to correlate these diversities, we expressed the different OppALl proteins in an oppA mutant with its native oppDFBC operon still functional. The ability of these engineered strains to transport peptides was compared with that of the corresponding wild-type strains. We reveal that, although OppALl was essential for peptide transport function, there was no correlation between the sequence of the binding protein and the specificity of peptide transport. These results suggest a role for the OppBCDF component in imposing the specificity of peptide transport in L. lactis. The bacterial strains used in this study are listed in TableI. L. lactis strains were stored at −80 °C in M17 broth (31Terzaghi B.E. Sandine W.E. Appl. Microbiol. 1975; 29: 807-813Crossref PubMed Google Scholar) containing 0.5% (w/v) glucose or lactose. E. coli strains were stored at −80 °C in LB broth (32Sambrook J. Fritsch E. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) containing 10% (v/v) glycerol and supplemented with chloramphenicol (5 mg/liter), erythromycin (2.5 mg/liter), or ampicillin (100 mg/liter) when required.Table IStrains and plasmids used in this studyStrain/plasmidCharacteristicsSource/Ref.E. coli NM522supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM) 5(rk− mk+) (F′ proAB, lacIqZΔM 15)Amersham Biosciences 8163NM522/pQEWThis work MC1022araD139Δ(ara,leu) 7697Δ(lacZ)M15 galU gal StrARef. 26Casadaban M.J. Cohen S.N. J. Mol. Biol. 1980; 138: 179-207Crossref PubMed Scopus (1751) Google Scholar 8159MC1022/pLEM1This work 8170MC1022/pLEM2This work 8179MC1022/pLEM3This work 8180MC1022/pLEM4This workL. lactis MG1363Plasmid-free, Lac−, Prt−, Opp+Ref. 27Gasson M.J. J. Bacteriol. 1983; 154: 1-9Crossref PubMed Google Scholar Wg2Wild-type, Lac+, Prt+, Opp+NIZO1-aNIZO, Netherland Dairy Research Institute (Ede, The Netherlands); INRA-URLGA, Unité de Recherches Laitières et Génétique Appliquée, Institut National de la Recherche Agronomique; NZDRI, New Zealand Dairy Research Institute (Palmerston North, New Zealand). IL1403Plasmid-free, Lac−, Prt−, Opp+Ref. 17Tynkkynen S. Buist G. Kunji E. Kok J. Poolman B. Venema G. Haandrikman A. J. Bacteriol. 1993; 175: 7523-7532Crossref PubMed Google Scholar CNRZ437Wild-type, Lac+, Prt+, Opp+INRA-URLGA CNRZ261Wild-type, Lac+, Prt+, Opp+INRA-URLGA E8Wild-type, Lac+, Prt+, Opp+NZDRI MG3+MG1363/pILpOL, pMG820, pLET5, Lac+, Prt−, Opp+This work AMP15MG1363, ΔoppA, Lac−, Prt−, Opp−Ref. 28Picon A. Kunji E.R.S. Lanfermeijer F.C. Konings W.N. Poolman B. J. Bacteriol. 2000; 182: 1600-1608Crossref PubMed Scopus (32) Google Scholar SL5145AMP15/pILpOL, pMG820, Lac+, Prt−, Opp−This work SL5146AMP15/pILpOL, pMG820, pLET5, Lac+, Prt−, Opp−This work SL5147AMP15/pILpOL, pMG820, pLEM1, Lac+, Prt−, Opp+This work SL5152AMP15/pILpOL, pMG820, pLEM2, Lac+, Prt−, Opp+This work SL5174AMP15/pILpOL, pMG820, pLEM3, Lac+, Prt−, Opp+This work SL5175AMP15/pILpOL, pMG820, pLEM4, Lac+, Prt−, Opp+This workPlasmid pQE30Vector of His6-tagged protein expressionQIAGEN S. A. pQEWpQE30 derivative carryingoppA gene from L. lactis Wg2This work pLET5E. coli-L. lactis expression vector; CmRRef. 29Wells J.M. Robinson K. Chamberlain L.M. Schofield K.M. Le Page R.W.F. Antonie Leeuwenhoek. 1996; 70: 317-330Crossref PubMed Scopus (124) Google Scholar pLEM1pLET5 derivative carryingoppA gene from L. lactis MG1363This work pLEM2pLET5 derivative carrying oppA gene fromL. lactis Wg2This work pLEM3pLET5 derivative carrying oppA gene from L. lactis IL1403This work pLEM4pLET5 derivative carrying oppA gene fromL. lactis CNRZ437This work pILpOL8.3-kb pAMβ1 derivative carrying gene coding for T7 RNA polymerase under control of lactococcal lac promoter; EryRRef. 29Wells J.M. Robinson K. Chamberlain L.M. Schofield K.M. Le Page R.W.F. Antonie Leeuwenhoek. 1996; 70: 317-330Crossref PubMed Scopus (124) Google Scholar pMG82023.7-kb derivative of pLP712 containing lacgenesRef. 30Maeda S. Gasson M.J. J. Gen. Microbiol. 1986; 132: 331-340PubMed Google Scholar1-a NIZO, Netherland Dairy Research Institute (Ede, The Netherlands); INRA-URLGA, Unité de Recherches Laitières et Génétique Appliquée, Institut National de la Recherche Agronomique; NZDRI, New Zealand Dairy Research Institute (Palmerston North, New Zealand). Open table in a new tab E. coli strains were grown with aeration at 37 °C in LB broth supplemented with the appropriate antibiotics when necessary.L. lactis wild-type strains were grown at 30 °C in M17 broth or in chemically defined medium (CDM) (33Poolman B. Konings W.N. J. Bacteriol. 1988; 170: 700-707Crossref PubMed Google Scholar). Engineered strains (SL5145, SL5146, SL5147, SL5152, SL5174, and SL5175) were grown in similar media containing both 0.5% (w/v) glucose and 0.25% (w/v) lactose and supplemented with erythromycin (2.5 mg/liter) and chloramphenicol (2.5 mg/liter). In some growth experiments, one of the essential amino acids (methionine, valine, histidine, glutamine, leucine, or isoleucine) was omitted from CDM and replaced with a peptide containing the omitted amino acid (final concentrations of 0.08, 0.28, 0.03, 0.27, 0.36, and 0.16 mmol/liter, respectively). Growth experiments were performed with an ultramicroplate reader (Bio-Tek Instruments, Inc., Winooski, VT) using 96-well sterile microplates. Each well contained 200 μl of culture medium and was inoculated with ∼107 colony-forming units/ml of an overnight culture. Prior to inoculation, the strain was washed twice with 50 mmol/liter KH2PO4/K2HPO4 (pH 6.9). The A600 was measured over 10 h every 30 min after gentle shaking. To prevent evaporation of the culture medium, each well was overlaid with sterile paraffin oil. The apparent growth rate is defined as the maximal slope of the semilogarithmic plot against time of A600 measurements. Total DNA of L. lactis strains was isolated from a 2-ml culture grown overnight in M17 broth. Cells were harvested by centrifugation at 8000 ×g for 10 min and resuspended for 2 h at 37 °C in 0.1 mol/liter Tris, 0.1 mol/liter EDTA, 25% (v/v) glucose, and 0.1 g/liter mutanolysin (pH 7.0). Cells were lysed by incubation for 30 min at 37 °C in 0.1 mol/liter Tris, 0.01 mol/liter EDTA, 0.5% (v/v) sarcosyl, 1 g/liter proteinase K, and 1.25 g/liter RNase. Proteins were removed by two successive 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol extractions and one 24:1 (v/v) chloroform/isoamyl alcohol extraction. The last supernatant volume was adjusted to 400 μl with Tris/EDTA/sarcosyl solution, and 50 μl of 3 mol/liter potassium acetate (pH 4.8) was added. DNA was precipitated with 1.2 ml of ice-cold ethanol and finally resuspended in 200 μl of 10 mmol/liter Tris buffer. The oppA genes from several lactococcal strains were amplified by PCR using primers oppstart (5′-ACACGCATGGACAAATTAAAAGTAACT-3′) and oppstop (5′-CGGGATCCAACTATTTGGTGGC-3′), designed according to the L. lactis SSL135 oppA sequence (17Tynkkynen S. Buist G. Kunji E. Kok J. Poolman B. Venema G. Haandrikman A. J. Bacteriol. 1993; 175: 7523-7532Crossref PubMed Google Scholar). In the case ofL. lactis IL1403, the primers were oppAstart* (5′-GGGCATGCAAAAATTAAAAGTAACT-3′) and oppAstop* (5′-GGATCCCTATTTGGTTGCCATCTTAT-3′) (16Bolotine A. Wincker P. Mauger S. Jaillon O. Malarme K. Weissenbach J. Ehrlich S.D. Sorokin A. Genome Res. 2001; 11: 731-753Crossref PubMed Scopus (964) Google Scholar). PCR products were restricted with SphI plus BamHI and cloned into expression plasmid pLET5 treated with the same restriction enzymes. The resulting hybrid plasmids (pLEM1 to pLEM4) were transferred into E. coli MC1022 (32Sambrook J. Fritsch E. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), and structure was confirmed by restriction digestion and DNA sequencing. After extraction from E. coli(34Birnboim H.W. Doly J. Nucleic Acids Res. 1979; 7: 1513-1523Crossref PubMed Scopus (9901) Google Scholar), the pLEM derivatives were transferred into L. lactis SL5145 by electroporation as previously described (35Wells J.M. Wilson P.W. Le Page R.W.F. J. Appl. Bacteriol. 1993; 74: 629-636Crossref PubMed Scopus (166) Google Scholar). Transformants were selected on M17 agar medium supplemented with 0.5% (w/v) lactose, 5 mg/liter erythromycin, and 5 mg/liter chloramphenicol. TheoppA open reading frame (not including the signal sequence codons) from L. lactis Wg2 was PCR-amplified using primers oppAsensHis2 (5′-CGCGGATCCAATCAAAGCTCAAGTACAAGTACA-3′) and oppArevHis1 (5′-CGGGGTACCCTATTTGGTGGCCAACTTAGC-3′). The PCR product was restricted with BamHI plus KpnI and cloned into the QIAexpress vector pQE30 (QIAGEN S. A., Courtaboeuf, France) restricted with the same enzymes. The resulting plasmid (pQEW) was then introduced into E. coliNM522, yielding E. coli 8163. Expression of the OppA-His6 protein was carried out in 500 ml of culture essentially as described by QIAGEN S. A. After removal of the culture medium, cells were resuspended in 10 mmol/liter Tris, 100 mmol/liter NaH2PO4, 8 mol/liter urea, 0.1% (v/v) Triton X-100, 20 mmol/liter β-mercaptoethanol, and 1 mg/liter lysozyme (pH 8.0). The cell suspension was incubated for 1 h at 25 °C with gentle agitation (200 rpm), and a clear cell-free extract was obtained by centrifugation at 12,000 × g for 15 min. Purification of the OppA-His6 protein was carried out under denaturing conditions by applying the cell-free extract to nickel-nitrilotriacetic acid resin (QIAGEN S. A.) and using an elution system of 10 mmol/liter Tris, 100 mmol/liter NaH2PO4, and 8 mol/liter urea according to the manufacturer's instructions. The OppA-His6-containing fractions, as determined by SDS-PAGE analysis, were dialyzed against sterile water and lyophilized. Anti-OppA antibodies were purified from the serum of an immunized rabbit (Valbex-Université Claude Bernard Lyon I, Villeurbanne, France) as described above, except that the resin was additionally washed with 40 ml of 50 mmol/liter Tris and 150 mmol/liter NaCl (pH 7.4) and then with 40 ml of 50 mmol/liter Tris and 1 mol/liter NaCl (pH 7.4). Antibodies were eluted during a 30-min incubation with 4 mol/liter MgCl2. The elution fractions were collected, dialyzed against sterile water, and lyophilized. L. lactis protein extracts were first separated by SDS-12% polyacrylamide gel electrophoresis and then electrotransferred onto nitrocellulose membrane (Schleicher & Schüll, Dassel, Germany). The OppA protein was detected by the method of Harlow and Lane (36Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar) using anti-OppA polyclonal antibodies (diluted 1:220), peroxidase-conjugated anti-rabbit IgG (diluted 1:4000; Sigma, Saint-Quentin Fallavier, France), and the BM chemiluminescence blotting substrate kit (Roche Molecular Biochemicals, Meylan, France). Milk proteins were precipitated with 1% (v/v) trifluoroacetic acid. After removal of the proteins by centrifugation at 10,000 × g for 10 min at 4 °C, the supernatant was ultrafiltered through a 3000-Da cutoff membrane (YM3, Amicon, Inc., Beverly, MA). Peptides were isolated by solid-phase extraction using reverse-phase cartridges (Sep-Pak C18, Waters Associates, Milford, MA). The peptides were separated at 40 °C by HPLC on a reverse-phase C18 column (Nucleosil (250 × 4.6 mm), Colochrom, Gagny, France) at a flow rate of 1 ml/min. Solvents A and B were 0.115% (v/v) trifluoroacetic acid and 0.1% (v/v) trifluoroacetic acid and 60% (v/v) acetonitrile in MilliQ water, respectively. A 5-min isocratic phase in solvent A was followed by a linear gradient of solvent B (0–60% within 40 min). The collected fractions were submitted to a second separation using 5 mmol/liter KH2PO4/K2HPO4 (pH 6.9) and 60% (v/v) acetonitrile in 5 mmol/liter KH2PO4/K2HPO4 (pH 6.9) as the solvents. The eluted peptides were collected, dried in a SpeedVac concentrator (Savant Instruments, Inc., Farmingdale, NY), resuspended in MilliQ water, and desalted using the first HPLC separation system (trifluoroacetic acid/acetonitrile). Purified peptides were identified by mass spectrometric analysis and N-terminal microsequencing. The transport assays were adapted from previously described procedures (2Kunji E.R.S. Hagting A. De Vries C.J. Juillard V. Haandrikman A.J. Poolman B. Konings W.N. J. Biol. Chem. 1995; 270: 1569-1574Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 37Kunji E.R.S. Mierau I. Poolman B. Konings W.N. Venema G. Kok J. Mol. Microbiol. 1996; 21: 123-131Crossref PubMed Scopus (33) Google Scholar). Cells were grown toA650 ∼ 0.8 in CDM containing free amino acids as the nitrogen source. Prior to transport assays, cells were washed twice with 50 mmol/liter KH2PO4/K2HPO4 (pH 6.9) and then de-energized for 30 min at 30 °C with 10 mmol/liter 2-deoxy-d-glucose (38Poolman B. Smid E.J. Konings W.N. J. Bacteriol. 1987; 169: 2755-2761Crossref PubMed Google Scholar). For each transport assay, cells (A650 = 1; corresponding to 0.2 g of cell protein/liter) were incubated for 5 min at 22 °C in the presence of 25 mmol/liter glucose and 2 mmol/liter MgSO4. When required, the serine proteinase inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (1 mmol/liter; Interchim, Montluçon, France) was added to the incubation mixture. We first verified that this inhibitor had no effect on transport process. Uptake was initiated by adding the peptide at a final concentration of 50 μmol/liter, unless otherwise stated. One-ml samples were taken, and cells were separated from the incubation medium by filtration using cellulose acetate filters (0.45-μm pore size; Schleicher & Schüll). Cells were subsequently washed twice with 2 ml of ice-cold KH2PO4/K2HPO4(50 mmol/liter) at pH 6.9. Peptide uptake was monitored by determining the intracellular concentration of free amino acids constituting the peptide under study, as previously described (2Kunji E.R.S. Hagting A. De Vries C.J. Juillard V. Haandrikman A.J. Poolman B. Konings W.N. J. Biol. Chem. 1995; 270: 1569-1574Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). It is worth mentioning that intact peptide could not be detected inside the cells due to the high rate of peptide hydrolysis by internal peptidases (37Kunji E.R.S. Mierau I. Poolman B. Konings W.N. Venema G. Kok J. Mol. Microbiol. 1996; 21: 123-131Crossref PubMed Scopus (33) Google Scholar). The amino acids were first derivatized with o-phthalaldehyde and then separated at 37 °C on a reverse-phase HPLC C18column (UptiSelect (250 × 4.6 mm), Interchim) at a flow rate of 1 ml/min. Solvent A was 50 mmol/liter sodium acetate (pH 5.7) and 3% (v/v) tetrahydrofuran, and solvent B was 95% (v/v) methanol and 5% (v/v) tetrahydrofuran. A 5-min isocratic phase in 18% (v/v) solvent B was followed by a linear gradient of solvent B (18–100% within 35 min). For detection of fluorescence, the excitation and emission wavelengths were 340 and 455 nm, respectively. The ability of lactococcal strains to bind peptide VGDE was estimated at 30 °C as follows. Concentrated de-energized cells (A650 ∼ 15) were incubated for 2 min (23Detmers F.J.M. Lanfermeijer F.C. Abele R. Jack R.W. Tampé R. Konings W.N. Poolman B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12487-12492Crossref PubMed Scopus (63) Google Scholar) in 50 mmol/liter KH2PO4/K2HPO4 (pH 6.5) containing 500 μmol/liter VGDE. Cells were collected on a 0.22-μm pore size filter (Schleicher & Schüll) and washed three times with ice-cold potassium phosphate buffer. The filter was then coated for 2 min with a solution of peptide YGGFL (500 μmol/liter) in potassium phosphate buffer. After removing cells by filtration, the peptides contained in the buffer were concentrated by solid-phase extraction using an anion cartridge exchanger (Accell Plus QMA, Waters Associates) and analyzed by HPLC as described above. Six strains of L. lactis were grown in CDM lacking an essential amino acid in the free form and supplied by a pure peptide. None of the strains was able to grow when the omitted amino acid was not replaced by a peptide. Twenty-five different peptides were selected on the basis of their various biochemical characteristics and their origin (TableII). Six of them were purified from milk. They were initially chosen because of their disappearance from milk after growth of some of the strains (data not shown).Table IIBiochemical features of peptides used in this studyAmino acid sequenceUsed as a source ofMolecular MasspIRelevant featureDaVAPGVal341.47.0VGDEVal418.44.1SGGFMMet496.65.6TGGFMMet510.65.6LPQYGln519.65.5Purified from milkDGGFMMet524.63.5MRFAMet524.711.3KGGFMMet537.79.0EGGFMMet538.63.8VRYLVal549.78.7Purified from milkYGGFLLeu555.66RGGFMMet565.710.0DMPIQAMet672.83.5β-Casein-(184–190)KMPIQAMet685.99.0Derivative of β-casein-(184–190)EMPIQAMet686.83.8Derivative of β-casein-(184–190)RMPIQAMet713.910.0Derivative of β-casein-(184–190)YMPIQAMet720.95.8Derivative of β-casein-(184–190)EQIVIRIle756.96.1Purified from milkYPFPGPIIle789.98.0TVYQHQKHis9038.3Purified from milkISQRYQKGln922.010Purified from milkAMKPWIQPKMet1098.410Purified from milkIARRHPYFLHis1172.411.6SFPWMESDVTMet1193.32.6DRVYIHPFHLHis1296.57.0RPKPQQFFGLMMet1347.711.7 Open table in a new tab Most peptides (18 of 25) were able to sustain growth of the six strains at a maximal rate in CDM deprived of one essential amino acid in the free form and provided in peptide form (Fig.1). Growth systematically corresponded to consumption of the peptide, as revealed by HPLC analysis of the culture medium (data not shown). Nevertheless, except for CNRZ261, none of the strains was able to use all the tested peptides as a source of amino acids. Five peptides (VGDE, DRVYIHPFHL, RPKPQQFFGLM, ISQRYQK, and LPQY) were differently consumed by the six L. lactis strains, suggesting that the strains under study do not share the same preferences for peptide utilization. Moreover, L. lactis Wg2 grew very poorly in the presence of the basic heptapeptide ISQRYQK as the source of Gln or Ile, whereas this strain grew at a maximal rate in the presence of the basic heptapeptide YPFPGPI (source of Ile) or TVYQHQK (source of Gln). This indicates that previous observations made with L. lactis MG1363, which indicated a preference for peptide utilization related to both the mass and the charge of the peptide (24Juillard V. Guillot A. Le Bars D. Gripon J.-C. Appl. Environ. Microbiol. 1998; 64: 1230-1236Crossref PubMed Google Scholar), cannot be extended to the L. lactisspecies. The peptides used for growth experiments were incubated with cell-free extracts. They were all cleaved at the amino acid level. The lack of growth of some strains in CDM was therefore not due to an inability of the cells to cleave the peptide intracellularly. The peptides were also incubated either in the presence of the PI-type proteinase PrtP released from L. lactisE8, CNRZ261, and Wg2 by incubation in a Ca2+-free buffer (39Laan H. Konings W.N. Appl. Environ. Microbiol. 1991; 57: 2586-2590Crossref PubMed Google Scholar) or in the presence of PIII-type PrtP anchored to resting L. lactis CNRZ437 cells (note that autoproteolysis of PIII-type PrtP affects its specificity) (40Flambard B. Juillard V. Appl. Environ. Microbiol. 2000; 66: 5134-5140Crossref PubMed Scopus (10) Google Scholar). Only one of them (RPKPQQFFGLM) was cleaved by PrtP. Other peptides were not hydrolyzed. This suggests that most (if not all) of the differences observed during growth experiments were not due to a difference in extracellular cleavage of the peptide by PrtP. Consequently, the most convenient hypothesis to explain the differences in growth is that the strains under study do not have identical oligopeptide transport capabilities. To ascertain this hypothesis, we tested the ability of four of the strains to transport three peptides that revealed a difference in preferences for peptide utilization between strains (VGDE, DRVYIHPFHL, and RPKPQQFFGLM). To prevent extracellular cleavage of RPKPQQFFGLM by PrtP, uptake was performed in the presence of 1 mm4-(2-aminoethyl)benzenesulfonyl fluoride. As expected, the ability of the strains to grow correlated with their ability to transport the peptides (Table III). By analyzing the presence of free Val or free Met in the external medium, we have experimentally excluded the possibility that differences in peptide uptake rates could be due to differences in amino acid efflux rates between strains. Despite the fact that VGDE ensured a maximal growth rate of L. lactis Wg" @default.
• W1978360923 created "2016-06-24" @default.
• W1978360923 creator A5004013927 @default.
• W1978360923 creator A5044835448 @default.
• W1978360923 creator A5050820294 @default.
• W1978360923 creator A5067859386 @default.
• W1978360923 creator A5089736835 @default.
• W1978360923 creator A5090278447 @default.
• W1978360923 date "2003-04-01" @default.
• W1978360923 modified "2023-10-18" @default.
• W1978360923 title "Diversity of Oligopeptide Transport Specificity in Lactococcus lactis Species" @default.
• W1978360923 cites W1507941369 @default.
• W1978360923 cites W1797512117 @default.
• W1978360923 cites W1823176675 @default.
• W1978360923 cites W1864840202 @default.
• W1978360923 cites W1970499058 @default.
• W1978360923 cites W1971289275 @default.
• W1978360923 cites W1975905184 @default.
• W1978360923 cites W1983517878 @default.
• W1978360923 cites W1984640298 @default.
• W1978360923 cites W1988988021 @default.
• W1978360923 cites W2009159314 @default.
• W1978360923 cites W2012501342 @default.
• W1978360923 cites W2012596360 @default.
• W1978360923 cites W2012823497 @default.
• W1978360923 cites W2013959608 @default.
• W1978360923 cites W2016888450 @default.
• W1978360923 cites W2019410656 @default.
• W1978360923 cites W2025013124 @default.
• W1978360923 cites W2026026226 @default.
• W1978360923 cites W2035100091 @default.
• W1978360923 cites W2041338197 @default.
• W1978360923 cites W2047005829 @default.
• W1978360923 cites W2060237749 @default.
• W1978360923 cites W2071773887 @default.
• W1978360923 cites W2077881869 @default.
• W1978360923 cites W2083361262 @default.
• W1978360923 cites W2103032054 @default.
• W1978360923 cites W2109327741 @default.
• W1978360923 cites W2113660908 @default.
• W1978360923 cites W2117648820 @default.
• W1978360923 cites W2123509606 @default.
• W1978360923 cites W2125920591 @default.
• W1978360923 cites W2127922557 @default.
• W1978360923 cites W2130073805 @default.
• W1978360923 cites W2132644314 @default.
• W1978360923 cites W2133827518 @default.
• W1978360923 cites W2139325759 @default.
• W1978360923 cites W2147655656 @default.
• W1978360923 cites W2163832720 @default.
• W1978360923 cites W2168772829 @default.
• W1978360923 cites W2169964279 @default.
• W1978360923 cites W2172068963 @default.
• W1978360923 doi "https://doi.org/10.1074/jbc.m212454200" @default.
• W1978360923 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12590143" @default.
• W1978360923 hasPublicationYear "2003" @default.
• W1978360923 type Work @default.
• W1978360923 sameAs 1978360923 @default.
• W1978360923 citedByCount "32" @default.
• W1978360923 countsByYear W19783609232012 @default.
• W1978360923 countsByYear W19783609232016 @default.
• W1978360923 countsByYear W19783609232017 @default.
• W1978360923 countsByYear W19783609232018 @default.
• W1978360923 countsByYear W19783609232019 @default.
• W1978360923 countsByYear W19783609232020 @default.
• W1978360923 countsByYear W19783609232022 @default.
• W1978360923 crossrefType "journal-article" @default.
• W1978360923 hasAuthorship W1978360923A5004013927 @default.
• W1978360923 hasAuthorship W1978360923A5044835448 @default.
• W1978360923 hasAuthorship W1978360923A5050820294 @default.
• W1978360923 hasAuthorship W1978360923A5067859386 @default.
• W1978360923 hasAuthorship W1978360923A5089736835 @default.
• W1978360923 hasAuthorship W1978360923A5090278447 @default.
• W1978360923 hasBestOaLocation W19783609231 @default.
• W1978360923 hasConcept C13935069 @default.
• W1978360923 hasConcept C144024400 @default.
• W1978360923 hasConcept C185592680 @default.
• W1978360923 hasConcept C19165224 @default.
• W1978360923 hasConcept C2775920511 @default.
• W1978360923 hasConcept C2779281246 @default.
• W1978360923 hasConcept C2780011460 @default.
• W1978360923 hasConcept C2780913471 @default.
• W1978360923 hasConcept C2781316041 @default.
• W1978360923 hasConcept C523546767 @default.
• W1978360923 hasConcept C54355233 @default.
• W1978360923 hasConcept C55493867 @default.
• W1978360923 hasConcept C70721500 @default.
• W1978360923 hasConcept C86803240 @default.
• W1978360923 hasConcept C89423630 @default.
• W1978360923 hasConceptScore W1978360923C13935069 @default.
• W1978360923 hasConceptScore W1978360923C144024400 @default.
• W1978360923 hasConceptScore W1978360923C185592680 @default.
• W1978360923 hasConceptScore W1978360923C19165224 @default.
• W1978360923 hasConceptScore W1978360923C2775920511 @default.
• W1978360923 hasConceptScore W1978360923C2779281246 @default.
• W1978360923 hasConceptScore W1978360923C2780011460 @default.
• W1978360923 hasConceptScore W1978360923C2780913471 @default.
• W1978360923 hasConceptScore W1978360923C2781316041 @default.

• next