Multiple-peptidase mutants of Lactococcus lactis are severely impaired in their ability to grow in milk.

TitleMultiple-peptidase mutants of Lactococcus lactis are severely impaired in their ability to grow in milk.
Publication TypeJournal Article
Year of Publication1996
AuthorsMierau, I, Kunji, ERS, Leenhouts, KJ, Hellendoorn, MA, Haandrikman, AJ, Poolman, B, Konings, WN, Venema, G, Kok, J
JournalJ Bacteriol
Volume178
Issue10
Pagination2794-803
Date Published1996 May
ISSN0021-9193
KeywordsAmino Acid Sequence, Amino Acids, Animals, Bacterial Proteins, Base Sequence, Biological Transport, Blotting, Southern, Blotting, Western, Carrier Proteins, Caseins, Cloning, Molecular, Endopeptidases, Lactococcus lactis, Lipoproteins, Milk, Molecular Sequence Data, Mutation, Peptides, Polymerase Chain Reaction, Sequence Deletion, Serine Endopeptidases
Abstract

To examine the contribution of peptidases to the growth of lactococcus lactis in milk, 16 single- and multiple-deletion mutants were constructed. In successive rounds of chromosomal gene replacement mutagenesis, up to all five of the following peptidase genes were inactivated (fivefold mutant): pepX, pepO, pepT, pepC, and pepN. Multiple mutations led to slower growth rates in milk, the general trend being that growth rates decreased when more peptidases were inactivated. The fivefold mutant grew more than 10 times more slowly in milk than the wild-type strain. In one of the fourfold mutants and in the fivefold mutant, the intracellular pools of amino acids were lower than those of the wild type, whereas peptides had accumulated inside the cell. No significant differences in the activities of the cell envelope-associated proteinase and of the oligopeptide transport system were observed. Also, the expression of the peptidases still present in the various mutants was not detectably affected. Thus, the lower growth rates can directly be attributed to the inability of the mutants to degrade casein-derived peptides. These results supply the first direct evidence for the functioning of lactococcal peptidases in the degradation of milk proteins. Furthermore, the study provides critical information about the relative importance of the peptidases for growth in milk, the order of events in the proteolytic pathway, and the regulation of its individual components.

Alternate JournalJ. Bacteriol.
Citation Key1143
PubMed ID8631666
PubMed Central IDPMC178013