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...

A chemically defined medium for the growth of Leuconostoc mesenteroides was developed. This medium contained lactose, Mn(sup2+), Mg(sup2+), 12 amino acids, eight vitamins, adenine, uracil, and Tween 80. We showed the beneficial effect of aerobic conditions on growth and that potassium phosphate (135 mM) is a suitable buffer. The growth rate in this medium was 0.85 (plusmn) 0.10 h(sup-1) for the six strains examined, and cell densities up to 3.5 x 10(sup9) CFU/ml were attained.

To explain the competition for nitrogenous nutrients observed in mixed strain cultures of Lactococcus lactis and Leuconostoc mesenteroides, the utilization of peptides as a source of essential amino acids for growth in a chemically defined medium was compared in 12 strains of dairy origin. Both species were multiple amino acid auxotrophs and harboured a large set of intracellular peptidases. Lactococcus lactis can use a wide variety of peptides up to 13 amino acid residues whereas Leuc. mesenteroides assimilated only shorter peptides containing up to seven amino acids. Growth was limited by the transport of peptides and not by their hydrolysis. The nutritional value of peptides varied with the strains and the composition of the peptides, L. lactis being advantaged over Leuc. mesenteroides.

Growth of Leuconostoc mesenteroides in milk was studied with respect to the proteinase and peptidase activities of the strains and their nutritional requirements. Ln. mesenteroides grew poorly in milk since none of the 14 strains studied exceeded 5×108 cfu/ml at the end of growth. Few strains displayed proteinase activity, and this did not contribute much to growth. The pattern of peptidase activities varied with the strain. Nitrogen starvation and a high requirement for Mn2+ were involved in the cause of growth deficiencies. Addition of amino acids, 50 mg Mg2+/l and 0·08–0·49 mg Mn2+/l stimulated growth of most leuconostoc strains up to 5×108 cfu/ml. Addition of 5 g glucose/l to milk containing amino acids, Mg2+ and Mn2+ or yeast extract stimulated the growth of seven and eight strains respectively up to 109 cfu/ml. No growth advantage was found in a N2 atmosphere. However, the addition of small amounts of Mn2+ to milk suppressed the inhibitory effect of aeration on the growth of Ln. mesenteroides UD23, suggesting a protective role of Mn2+ against O2 toxicity.

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