Reconstruction of the proteolytic pathway for use of β‐casein by Lactococcus lactis

Abstract

SummaryAmino acid auxotrophous bacteria such as Lactococcus lactis use proteins as a source of amino acids. For this process, they possess a complex proteolytic system to degrade the protein(s) and to transport the degradation products into the cell. We have been able to dissect the various steps of the pathway by deleting one or more genes encoding key enzymes/components of the system and using mass spectrometry to analyse the complex peptide mixtures. This approach revealed in detail how L. lactis liberates the required amino acids from ␤-casein, the major component of the lactococcal diet. Mutants containing the extracellular proteinase PrtP, but lacking the oligopeptide transport system Opp and the autolysin AcmA, were used to determine the proteinase specificity in vivo. To identify the substrates of Opp present in the casein hydrolysate, the PrtP-generated peptide pool was offered to mutants lacking the proteinase, but containing Opp, and the disappearance of peptides from the medium as well as the intracellular accumulation of amino acids and peptides was monitored in peptidase-proficient and fivefold peptidase-deficient genetic backgrounds. The results are unambiguous and firmly establish that (i) the carboxyl-terminal end of ␤-casein is degraded preferentially despite the broad specificity of the proteinase; (ii) peptides smaller than five residues are not formed in vivo ; (iii) use of oligopeptides of 5-10 residues becomes only possible after uptake via Opp; (iv) only a few (10-14) of the peptides generated by PrtP are actually used, even though the system facilitates the transport of oligopeptides up to at least 10 residues. The technology described here allows us to monitor the fate of individual peptides in complex mixtures and is applicable to other proteolytic systems.

Kinetics and Substrate Specificity of Membrane-Reconstituted Peptide Transporter DtpT of Lactococcus lactis

“…For high-affinity interaction with DtpT, the peptides need to have free amino and carboxyl termini, amino acids in the L configuration, and trans-peptide bonds. Comparison of the specificity of DtpT with that of the eukaryotic homologues PepT 1 and PepT 2 shows that the bacterial transporter is more restrictive in its substrate recognition.Studies of the transport of peptides across the cell membrane of Lactococcus lactis have established that the organism uses at least three distinct peptide transport systems (5,17,20,21,32,35,36; Y. Sanz Herranz, F. C. Lanfermeijer, W. N. Konings, and B. Poolman, submitted for publication). In addition to the ATP-dependent ATP-binding cassette (ABC)-type of oligo-and di-and/or tripeptide transport systems, Opp and DtpP, respectively, L. lactis possesses a proton motive force-driven di-and/or tripeptide transporter DtpT.…”

Peptides and ATP binding cassette peptide transporters

“…In case of L. lactis it has been shown that β-casein is degraded to peptides of five to 30 amino acid residues, and a substantial fraction of these is imported into the cell via Opp. It has been shown that Opp of L. lactis accepts peptides in the range of five to ten residues when it is supplied with a complex mixture of β-casein-derived peptides [28]. This surprising finding was corroborated later on in studies with individual peptides of various lengths, where it was shown that peptides up to at least 18 residues are transported [10].…”

Lactococcus lactis LM0230 contains a single aminotransferase involved in aspartate biosynthesis, which is essential for growth in milk

“…During six growth curve replicates, the strain which lacked both the wild-type chromosomal and plasmid copy of aspC never acidified milk to more than 0n20 pH units below that of the blank and did not grow at a detectable rate. As oligopeptides liberated from caseins by the lactococcal PrtP serve as the major nitrogen source for L. lactis in milk (Juillard et al, 1995a), and L. lactis can apparently transport and hydrolyse at least one Asp-containing peptide from β-casein (Kunji et al, 1998), the inability of JLS400(pJK550, pTRKL2) to rapidly grow in or acidify milk suggests L. lactis is unable to liberate an adequate level of Asp or Asn from milk caseins to support optimal growth. As Asp is the biosynthetic precursor for Asn, threonine, lysine and methionine, as well as pyrimidine and purine nucleotides, Asp biosynthesis may also be essential for supplying the cell with sufficient levels of these biological compounds.…”

“…In vitro data suggests that lactococci can obtain all amino acids through hydrolysis and transport of β-casein-derived oligopeptides (Juillard et al, 1995b). However, more recent in vivo studies have shown that lactococci utilize only a limited number of oligopeptides derived from the C-terminal end of β-casein (Kunji et al, 1998).…”