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

Abstract

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-␤-chloro-L-alanine has been used to select for a di-tripeptide transport-negative mutant with the ⌬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.For optimal growth in milk, lactococci depend on the presence of a proteolytic system which consists of a cell envelopelocated proteinase, several peptidases, and amino acid and peptide transport systems (20). At present, two peptide transport systems have been characterized for Lactococcus lactis (6,10,11,29). The oligopeptide transport system (Opp) mediates the ATP-driven transport of peptides with four to at least eight residues. It plays a central role in the proteolytic pathway of L. lactis, as it is essential for the accumulation of all ␤-caseinderived amino acids (10). The level of activity in the Opp system is sufficiently high to support maximal growth rates on ␤-casein, provided that leucine and histidine are present in the medium as free amino acids. The di-and tripeptide transport system (DtpT) is unique among bacterial peptide transporters, as it is encoded by a single gene and uses the proton motive force to drive the transport of relatively hydrophilic di-and tripeptides (6). Spontaneous mutations which inactivate the dtpT gene lead to defective growth of L. lactis in chemically defined medium (CDM) supplemented with a mixture of caseins as the sole source of nitrogen (24).On the basis of the observation that the transport of some di-and tripeptides is totally abolished in alanyl-␤-chloroalanine-resistant mutants of L. lactis whereas other peptides are still taken up at significant rates (26), the utilization of di-and tripeptides in mutants lacking either Opp or DtpT or both peptide transport systems was investigated. In this report, we present evidence for a third peptide transport system in L. lactis with specificity for relatively hydrophobic di-and tripeptides. MATERIALS AND METHODSBacterial strains, culture conditions, and growth media. L. lactis subsp. lactis MG1363 wild-type and isogenic mutants, i.e., the di-and tripeptide transport mut...

Utilisation des sources d'azote du lait par Lactococcus lactis

“…Il a récemment été mis en évi-dence et caractérisé (Foucaud et al, 1995 Foucaud et al, 1995). Transport of peptides by lactococcus lactis (from Foucaud et al, 1995 L'obtention récente de mutants dérivés d'une souche dépourvue de protéase de paroi (souche Prr), et déficients pour l'un ou l'autre des systèmes de transport des peptides, a permis d'estimer le rôle joué par les peptides initialement présents dans le lait lors de la croissance (tableau IV). L'inactivation des systèmes de transport des di-et tripeptides (DtpT seul, ou DtpT et DtpP) n'affecte pas le niveau cellulaire en fin de culture.…”

“…Cette diminution du taux de croissance n'est pas observée lorsque seul le système DtpT est inactivé (Juillard et al, 1995a L'analyse du contenu peptidique du lait avant et après croissance de ces différentes souches est en parfait accord avec ces résultats (Juillard et al, 1995a Juillard et al, 1995a;Foucaud et al, unpublished results a Les phénotypes DtpT -et Opp-ont respectivement été obtenus par délétion du gène dtpT (Hagting et al, 1994) ou par disruption du gène oppA (Tynkkynen et al, 1993 (Hagting et al, 1994) and from the disruption of the oppA gene (Tynkkynen et al, 1993), respectively. Both L lactis CF310 and CFS62 strains were selected for resistance towards the toxic peptide Phe-[3chloro-Ala from L lactis AG300 (Foucaud et al, 1995) andL lactis CV4…”

Membrane Topology of the Di- and Tripeptide Transport Protein ofLactococcus lactis

“…Transport of the more hydrophobic di-and tripeptides is catalyzed by another di-and tripeptide transport system DtpP (Foucaud et al, 1995). This transport is ATPdriven, like the uptake of peptides of more than three amino acid residues via the oligopeptide transport system Opp .…”

The Lactococcus lactis CodY Regulon

“…These peptides of various sizes, and free amino acids, can then be taken up into the cell by various transport systems, e.g. the oligopeptide transport system Opp (4) and the di-and tripeptide transport systems DtpP and DtpT (7,8). Once inside, the peptides are degraded further by either endopeptidases (e.g.…”