Identification and functional analysis of two oligopeptide transporters supporting the growth of Lacticaseibacillus paracasei strain Shirota in milk

Oligopeptide transporters are important proteins in several lactic acid bacteria (LAB) that facilitate the transport of oligopeptides, the primary nitrogen source for growth in milk. Although the proteolytic mechanisms are well understood in some LAB species, limited research has been conducted on the peptide transport systems of <i>Lacticaseibacillus paracasei</i> (formerly <i>Lactobacillus casei</i>) strain Shirota (LcS), particularly its oligopeptide transporters. This study investigated the nitrogen uptake mechanism of LcS, a probiotic lactic acid bacterium, by generating gene knockout (KO) strains of two oligopeptide transporters, Opp_Lc and Dpp_Lc. Consequently, the disruption of these genes eliminated the ability of the bacterium to grow in milk, identifying Opp_Lc and Dpp_Lc as the primary oligopeptide transporters in LcS. Growth in a leucine-free chemically defined medium with a Leu-containing peptide as the sole nitrogen source indicated that Opp_Lc and Dpp_Lc transport peptides of 4-8 and 3-7 residues, respectively. To our knowledge, this study provides the first experimental evidence of oligopeptide transporters in <i>Lactobacilli</i> capable of transporting peptides up to eight residues long. Analysis of KO strains targeting OppA₁ or DppA₁ to identify other oligopeptide-binding proteins (OBPs) within each oligopeptide transporter operon that may influence substrate specificity revealed that OppA₁ is the only OBPs in Opp_Lc. However, DppA₂ and DppA₃, encoded at chromosomal locations distant from the Dpp_Lc operon, may function as subunits constituting Dpp_Lc and DppA₁. These findings enhance our understanding of nitrogen source utilization in lactobacilli and might inform future strategies to optimize nitrogen sources for LcS and improve culture technology for LcS-based products.