MULTIPLE-PEPTIDASE MUTANTS OF LACTOCOCCUS-LACTIS ARE SEVERELY IMPAIRED IN THEIR ABILITY TO GROW IN MILK

To examine the contribution of peptidases to the growth of Lactococus lactis in milk, 16 single- and multiple-deletion mutants were construc ted. In successive rounds of chromosomal gene replacement mutagenesis, up to all five of the following peptidase genes were inactivated (fiv efold mutant): pepX pepO, pepT, pepC, and pepN. Multiple mutations led to slower growth rates in milk, the general trend being that growth r ates decreased when more peptidases were inactivated. The fivefold mut ant grew more than 10 times more slowly in milk than the wild-type str ain. In one of the fourfold mutants and in the fivefold mutant, the in tracellular pools of amino acids were lower than those of the wild typ e, whereas peptides had accumulated inside the cell. No significant di fferences in the activities of the cell envelope associated proteinase and of the oligopeptide transport system were observed. Also, the exp ression of the peptidases still present in the various mutants was not detectably affected, Thus, the lower growth rates can directly be att ributed to the inability of the mutants to degrade casein-derived pept ides. These results supply the first direct evidence for the functioni ng of lactococcal peptidases in the degradation of milk proteins. Furt hermore, the study provides critical information about the relative im portance of the peptidases for growth in milk, the order of events in the proteolytic pathway, and the regulation of its individual componen ts.