Diversity of Oligopeptide Transport Specificity in Lactococcus lactis Species

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. 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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 between the sequence of the binding protein and the specificity of peptide transport. results a role for the in the specificity of peptide transport in L. lactis. The strains used in this are in L. lactis strains were at in Appl. Microbiol. PubMed Google Scholar) containing E. coli strains were at in J. E. Scholar) containing and with and used in this coli S.N. J. Mol. Biol. PubMed Scopus Google lactis J. Bacteriol. PubMed Google The S. Buist G. Kunji E. Kok J. Poolman B. Venema G. Haandrikman A. J. Bacteriol. 1993; 175: 7523-7532Crossref PubMed Google A. Kunji E.R.S. Lanfermeijer F.C. Konings W.N. Poolman B. J. Bacteriol. 2000; PubMed Scopus Google of protein S. A. gene from L. lactis lactis J.M. Robinson K. Le 1996; PubMed Scopus Google gene from L. lactis oppA gene lactis oppA gene from L. lactis oppA gene lactis gene for of J.M. Robinson K. Le 1996; PubMed Scopus Google of containing S. J. Microbiol. Google The in a new E. coli strains were with at in with the lactis wild-type strains were at in in chemically defined medium B. Konings W.N. J. Bacteriol. PubMed Google Scholar). strains and were in similar containing both and and with and In some growth one of the essential amino acids was from and with a peptide containing the amino acid of and experiments were with an Each well of medium and was with of an to the strain was with The was of the well was with The growth rate is defined as the of the of of L. lactis strains was from a in were by at for and for at in and were by for at in and were by two and one The was to with and of was was with of and in of The oppA genes from several strains were by and to the L. lactis 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 ofL. lactis the were and (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). were with and cloned with the The to were E. coli J. E. Scholar), and was by and from E. J. Res. PubMed Scopus Google Scholar), the were L. lactis by as described J.M. Le J. Appl. Bacteriol. 1993; PubMed Scopus Google Scholar). were medium with and the sequence from L. lactis was and The was with and cloned the S. with the The was E. E. coli of the protein was in of as described by S. A. of the were in and The cell was for at with and a was by at for of the protein was by the to acid S. and an system of and to the The as by were and were from the of an as described that the was with of and and with of and were during a with The were and L. lactis protein were by and membrane The OppA protein was by the of and E. D. Scholar) and the substrate proteins were with of the proteins by at for at the was a membrane were by The peptides were at by a at a rate of and were acid and acid and in in was by a of The were to a and in as the The peptides were in a in and the system peptides were by and N-terminal The transport were from described 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, E.R.S. Poolman B. Konings W.N. Venema G. Kok J. Mol. Microbiol. 1996; 21: PubMed Scopus Google Scholar). were in containing amino acids as the to transport were with and for at with B. Smid E.J. Konings W.N. J. Bacteriol. 1987; 169: PubMed Google Scholar). transport corresponding to of cell were for at in the of and the was to the We that this transport process. was by the peptide at a of were and were from the medium by were with of at uptake was by the intracellular of amino acids the peptide as described 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 that peptide not be the to the high rate of peptide hydrolysis by E.R.S. Poolman B. Konings W.N. Venema G. Kok J. Mol. Microbiol. 1996; 21: PubMed Scopus Google Scholar). The amino acids were with and at a at a rate of was and and was and in was by a of 35 of the and were and The ability of strains to bind peptide was at as were for (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 containing were a and with The was for with a of peptide in by the peptides in the were by an and by as described Six strains of L. lactis were in an essential amino acid in the and by a of the strains was able to the amino acid was not by a different peptides were the of their and their Six of were from milk. were of their from growth of some of the strains not of peptides used in this acid as a from from of of of of from from from from in a new peptides of were able to growth of the strains at a rate in of one essential amino acid in the and in peptide to consumption of the as revealed by of the medium not Nevertheless, for of the strains was able to the tested peptides as a of amino peptides and were by the L. lactis that the strains not the preferences for peptide L. lactis in the of the basic as the of whereas this strain at a rate in the of the basic of of This that with L. lactis which a for peptide to both the and the of the peptide (24Juillard V. Guillot A. Le Bars D. Gripon J.-C. Appl. Environ. Microbiol. 1998; 64: 1230-1236Crossref PubMed Google Scholar), be to the L. The peptides used for growth experiments were with were at the amino acid level. The of growth of some strains in was not to an of the to the peptide The peptides were in the of the from L. and by in a Konings W.N. Appl. Environ. Microbiol. 1991; PubMed Google Scholar) in the of anchored to L. lactis that of its B. Juillard V. Appl. Environ. Microbiol. 2000; PubMed Scopus Google Scholar). one of was by peptides were not This that not of the differences during growth experiments were not to a in extracellular of the peptide by the to the differences in growth is that the strains not oligopeptide transport this we tested the ability of four of the strains to transport peptides that revealed a in preferences for peptide between strains and extracellular of by uptake was in the of the ability of the strains to with their ability to transport the peptides the of in the we the that differences in peptide uptake be to differences in amino acid between strains. Despite the fact that a growth rate of L. lactis a in the initial rate of uptake was between the two strains. the not at a rate in the of although its initial rate of uptake was higher than that of This that the rate of peptide transport not the growth at rate of peptide transport by wild-type L. lactis used for transport experiments were for and for and The amino acids used to the rate of uptake of and were and The of two is the of was in a new used for transport experiments were for and for and The amino acids used to the rate of uptake of and were and The of two is the of was The binding protein is generally considered to be for the specificity of peptide transport in ATP-binding cassette (19Lanfermeijer F.C. Picon A. Konings W.N. Poolman B. Biochemistry. 1999; 38: 14440-14450Crossref PubMed Scopus (47) Google Scholar). The oppA genes lactis strains and were cloned and of the amino acid in the and revealed some differences at the amino acid The OppA sequence of MG1363 was to that of but from of and by and In an attempt to the differences in peptide transport to the in the OppA amino acid the from four different strains were the strain A. Kunji E.R.S. Lanfermeijer F.C. Konings W.N. Poolman B. J. Bacteriol. 2000; PubMed Scopus Google Scholar). The oppA genes were cloned the of the J.M. Robinson K. Le 1996; PubMed Scopus Google Scholar). the of the two and were in the strain, L. lactis strains and genes from and of and were in MG1363 and and The growth and in of strains and were similar and The of OppA expressed by and in the medium containing as the as the J.M. Robinson K. Le 1996; PubMed Scopus Google Scholar), and and were in the range and in than that by L. lactis MG1363 as revealed by a significant differences in the uptake of the peptide by MG1363 and be whereas was to transport peptides. the of peptide transport by matched of L. lactis and the oppA genes from and the preferences for peptide as MG1363 strains and were to in containing as the of whereas the lactis wild-type strains and the growth of and MG1363 in containing as the of were although and were to this peptide as the of It is lactis strains and are strains and to peptides in the medium to transport. experiments that L. lactis strains and transported the peptides their of different OppA proteins The ability of the engineered strains to transport specific peptides was to that of of uptake were than with the wild-type This was to a of the binding protein in the engineered strains compared with MG1363 The of with the binding protein from L. lactis strain the substrate specificity of L. lactis MG1363 than that of the OppA rate of peptide transport by engineered L. lactis used for transport experiments were for and for and The amino acids used to the rate of uptake of and were and The of two is the of was not in a new used for transport experiments were for and for and The amino acids used to the rate of uptake of and were and The of two is the of was not The ability of the binding protein OppA from L. lactis strain MG1363 to bind in non-transported peptides was were in the of the non-transported peptide were in the of The of from OppA by was by the to its peptide by be was from the only of were L. lactis that this strain not bind a significant of during the In L. lactis MG1363 of of protein in the of of S.D. results demonstrate the ability of L. lactis MG1363 OppA to interact in with a non-transported of substrate specificity in peptide transport by L. lactis were only one strain, MG1363 (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, V. Guillot A. Le Bars D. Gripon J.-C. Appl. Environ. Microbiol. 1998; 64: 1230-1236Crossref PubMed Google Scholar). The results demonstrated that the substrate specificity of L. lactis strain MG1363 is compared with Gram-negative bacteria coli and S. typhimurium (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, 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 present evidence for variability in the ability of L. lactis strains to peptides as a of amino acids during MG1363 was to the as the of This is in with the preferences for peptide by this strain, of peptides with low molecular (24Juillard V. Guillot A. Le Bars D. Gripon J.-C. Appl. Environ. Microbiol. 1998; 64: 1230-1236Crossref PubMed Google Scholar). this peptide was by the strains the MG1363 preferences are not of the We were able to four strains the of their to several peptides. This variability in peptide consumption from a in the ability of the strains to transport peptides. The specificity of peptide transport by L. lactis strain MG1363 not that of the strains. is that the L. lactis shows a biodiversity in oligopeptide transport specificity. by L. lactis the of the functional oligopeptide transport system that the binding protein OppA the specificity of the Opp system A. Kunji E.R.S. Lanfermeijer F.C. Konings W.N. Poolman B. J. Bacteriol. 2000; PubMed Scopus Google Scholar, Tame J.R.H. Dodson E.J. Wilkinson A.J. Biochemistry. 1997; PubMed Scopus Google Scholar). a mutant was to be to transport peptides, and of OppA the specificity of the protein for peptides A. Kunji E.R.S. Lanfermeijer F.C. Konings W.N. Poolman B. J. Bacteriol. 2000; PubMed Scopus Google Scholar). The biodiversity in peptide transport specificity in was used as a tool to unravel the role of the binding protein OppALl in the substrate specificity of L. lactis. of the OppALl of the four strains some amino acid to these were for the we cloned and expressed genes of the four strains in the lactis A. Kunji E.R.S. Lanfermeijer F.C. Konings W.N. Poolman B. J. Bacteriol. 2000; PubMed Scopus Google Scholar). by different oppA genes four functional Opp transporters. This that of the in the different OppALl proteins donation of the substrate from the binding protein to the transmembrane of The substrate specificity of the four engineered strains was found to be to that of L. The substrate specificity of the L. lactis strains and not that of the corresponding wild-type strains and results the role of OppALl in the specificity of peptide transport. a peptide is not by it not be transported by the Opp In this OppALl might be considered as one determinant of peptide transport specificity. Nevertheless, the specificity of peptide binding by OppALl is broad (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). The of strains to that the of to transport specific peptides was not to peptide by In the specificity of peptide transport the ability to transport specific peptides is not The broad binding specificity of it from the specificity determinant of oligopeptide transport. is that, although OppALl is for peptide transport, it not its specificity. results this experiments that the was able to with the protein of L. lactis This was in with the by the transport of a peptide by L. and J. Appl. in This that a peptide bind to OppALl it is not transported by the Opp results the specificity of peptide uptake lactis is not by The for oligopeptide transport by L. a (i) binding of the substrate to the of the binding (ii) of the binding in the of the (iii) of the substrate from the binding protein to the transmembrane and (iv) of the substrate the membrane (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, F.C. F.J.M. Konings W.N. Poolman B. J. 2000; PubMed Scopus Google Scholar). The peptide was by the L. lactis strains and at different although was in the range for the strains This that the of transport, peptide binding by is not a for the transport process. It is in with that demonstrated that the rate of transport is by the of peptide donation from the binding protein to the complex (19Lanfermeijer F.C. Picon A. Konings W.N. Poolman B. Biochemistry. 1999; 38: 14440-14450Crossref PubMed Scopus (47) Google Scholar). In this the binding protein as a that the from to the medium J. Bacteriol. 2002; PubMed Scopus Google Scholar). of the peptide binding be to a the transmembrane complex to the ATP that results in an in the transporter affinity for ATP and to the of the and the of the substrate from the binding proteins and been described as in several be by in the binding protein and of the complex Infect. Immun. PubMed Google Scholar, Pearce J. Infect. Immun. 1995; PubMed Google Scholar). the is by the binding protein Infect. Immun. PubMed Google Scholar). is that the transmembrane complex OppBC as a It is that the sequences variability in the and sequences (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, 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). This sequence variability might the transport variability L. lactis strains. this is it to be the transmembrane complex in the specificity of the oligopeptide transport of specific peptides from the of the of the transmembrane complex with the binding protein with specific results that the specificity of oligopeptide transport inL. lactis is by at two The oligopeptides and the transport but is the the transmembrane and at the of involved in oligopeptide transport specificity in L. lactis. We A. for and of the