Peptide uptake is essential for growth of Lactococcus lactis on the milk protein casein

Abstract

The chlorated dipeptide L-alanyl-j-chloro-L-alanine (diACA) is very toxic for Lactococcus lactis. Spontaneous mutants resistant to the dipeptide were isolated from plates. The presence and activities of cell wall-associated proteinase, different peptidases in cell extracts, amino acid transport systems, and di-and oligopeptide transport systems were examined and compared in a diACA-resistant mutant and the wild type. Only the rates of di-and tripeptide transport were found to be significantly reduced in the diACA-resistant mutant ofL. lactis ML3. Since all other characteristics of this mutant were comparable to those of the wild type, the diACA-resistant mutant is most likely deficient in di-and tripeptide transport. Uptake of di-and tripeptides by L. lactis ML3 was found to be mainly mediated by one peptide transport system. The peptide transport-deficient mutant was found to be unable to grow on a chemically defined medium supplemented with casein as the sole nitrogen source, whereas growth could be restored by the addition of amino acids. These results indicate that peptide transport in L. lactis ML3 is an essential component in the process of casein utilization during growth in milk.In the dairy industry, lactococci (previously named group N lactic streptococci [19]) are commonly used as starter cultures in milk fermentations. These gram-positive bacteria are very fastidious and require exogenous sources of nucleotides, vitamins, and amino acids (16). Several amino acids are either stimulatory or essential for lactococcal growth (5, 16). The concentrations of free amino acids in milk are, however, too low to support optimal starter growth (22). For rapid acid production in milk fermentations, these organisms possess proteinases and a number of peptidases (see reference 22 for a recent review) which hydrolyze the milk protein casein and supply the cells with the required amino acids. Most strains of Lactococcus lactis can utilize amino acids and peptides with up to approximately four to six residues to satisfy their growth requirements (9,18,21,24).Hugenholtz et al. (5) showed that the rate of growth of L. lactis subsp. cremoris in milk is determined by the rate of casein hydrolysis and that casein limitation leads to a limitation of amino acids. These results raised the question of whether the casein-derived limiting amino acids were supplied as free amino acids or as small peptides.Different studies were undertaken to elucidate the actual pathway of casein utilization by lactococci. Proteolytic attack of bovine P-casein by the cell wall proteinases of L. lactis (12) and L. lactis subsp. cremoris (25, 26) can occur at seven cleavage positions at the C-terminal stretch of the milk protein. Proteolysis thus results in a mixture of peptide fragments which have to be further degraded by extracellular peptidases before they can pass through the cytoplasmic membrane. The information available about the extracellular breakdown of these peptide fragments is limited. It is still not clear how many extracellular pept...

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

“…The accumulation of the amino acids present in the peptides was clearly not due to external breakdown of the peptides followed by transport of the amino acids since (i) no free amino acids were detected in the external medium, (ii) the accumulation of amino acids was observed only with transportable peptides, the nontransported peptides were hydrolyzed upon permeabilization of the cells, and (iii) the di-and tripeptide transport activity was completely lost in the ⌬dtpT FCA r mutant, the peptidase activities in the wildtype strain and in the ⌬dtpT FCA r mutant were similar. DtpT and the newly described DtpP system have overlapping specificities for di-and tripeptides but do not transport amino acids or oligopeptides (Table 2 and results not shown) (11,25,26). The DtpT carrier transports relatively hydrophilic di-and tripeptides such as Glu-Val, Gly-Asp, Met-Asp, Ala-Gly, and Pro-Gly (6,26; also this study), whereas DtpP transports a variety of structurally different di-and tripeptides, with an apparent preference for hydrophobic (branched-chain) residues.…”

“…Toxic-peptide-resistant mutants were isolated from L. lactis AG300 according to the method of Smid et al (26). Approximately 10 8 CFU of an exponentially growing CDM culture were spread on a 1.2% (wt/vol) agar plate containing a complete amino acid mixture.…”

“…As expected L. lactis MG1363 and VS772 (OppA Ϫ ) can satisfy their requirements for essential amino acids by utilization of all diand tripeptides except for Ile-Arg (26) and Gly-His-Gly. Surprisingly, L. lactis AG300, which lacks the di-and tripeptide transport system DtpT, can utilize the same set of peptides except for Gly-Leu.…”

“…The toxicity of the peptide analog L-alanyl-␤-chloro-L-alanine has previously been used to isolate peptide transport mutants with DtpT Ϫ phenotypes (26). Since the putative second di-and tripeptide transport system has a specificity for more-hydrophobic peptides, we tested the toxicity of the peptide analog FCA on confluently plated L. lactis AG300 (⌬dtpT) cultures as described in Materials and Methods.…”

“…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.…”