The specific oligopeptide transport system Opp is essential for growth of Lactococcus lactis in milk. We examined the biodiversity of oligopeptide transport specificity in the L. lactis species. Six strains were tested for (i) consumption of peptides during growth in a chemically defined medium and (ii) their ability to transport these peptides. Each strain demonstrated some specific preferences for peptide utilization, which matched the specificity of peptide transport. Sequencing of the binding protein OppA in some strains revealed minor differences at the amino acid level. The differences in specificity were used as a tool to unravel the role of the binding protein in transport specificity. The genes encoding OppA in four strains were cloned and expressed in L. lactis MG1363 deleted for its oppA gene. The substrate specificity of these engineered strains was found to be similar to that of the L. lactis MG1363 parental strain, whichever oppA gene was expressed. In situ binding experiments demonstrated the ability of OppA to interact with non-transported peptides. Taken together, these results provide evidence for a new concept. Despite that fact that OppA is essential for peptide transport, it is not the (main) determinant of peptide transport specificity in L. lactis.

Growth of Lactococcus lactis in milk depends on the utilization of extracellular peptides. Up to now, oligopeptide uptake was thought to be due only to the ABC transporter Opp. Nevertheless, analysis of several Opp-deficient L. lactis strains revealed the implication of a second oligopeptide ABC transporter, the so-called Opt system. Both transporters are expressed in wild-type strains such as L. lactis SK11 and Wg2, whereas the plasmid-free strains MG1363 and IL-1403 synthesize only Opp and Opt, respectively. The Opt system displays significant differences from the lactococcal Opp system, which made Opt much more closely related to the oligopeptide transporters of streptococci than to the lactococcal Opp system: (i) genetic organization, (ii) peptide uptake specificity, and (iii) presence of two oligopeptide-binding proteins, OptS and OptA. The fact that only OptA is required for nutrition calls into question the function of the second oligopeptide binding protein (Opts). Sequence analysis of oligopeptide-binding proteins from different bacteria prompted us to propose a classification of these proteins in three distinct groups, differentiated by the presence (or not) of precisely located extensions.The ABC transporter family is characterized by a high affinity for their substrates, and it uses ATP hydrolysis to drive a unidirectional accumulation of solutes into the bacterial cytoplasm. A structural similarity of five proteins is generally observed (10). The substrate-binding protein specifically captures the substrate and is therefore considered one of the determinants of transport specificity (5). The binding protein is located in the periplasm of gram-negative bacteria. In gram-positive bacteria, it is present as a lipoprotein anchored to the cell membrane via an N-terminal moiety. The structure of the protein consists of three distinct domains. Two distinct lobes are connected by a flexible hinge (29), which allows two different conformations: an open, unliganded form with a high affinity for the substrate, and a closed, liganded state, in which the substrate is entrapped into the binding protein (the socalled Venus flytrap model). The closed liganded state is characterized by a low affinity for the substrate. The closed liganded binding protein delivers the substrate to the translocon, which consists of two integral transmembrane proteins and two ATP-binding proteins located on the cytoplasmic side of the membrane. According to a recent model (38), a consequence of the substrate binding would be the transmission of a signal to the ATP subunits of the ABC system. This signal results in an increased affinity of the ATP-binding proteins for ATP, which in turn leads to the opening of the transmembrane channel and the simultaneous release of the solute from the binding protein to the opened pore (6).In gram-positive bacteria, uptake of peptides by ABC transport systems is involved in nitrogen nutrition of the organism, as has been well established for lactic acid bacteria, for instance (11,22), or in the regula...

Aim:To study the effect of casein-derived peptides, accumulated during growth of Lactococcus lactis in milk, on its oligopeptide transport (Opp) function. Methods and Results: This effect was estimated by analysing the ability of casein-derived peptides to compete for the transport of a reporter peptide by whole L. lactis cells. The transport of the reported peptide was monitored by determining the intracellular concentrations of the corresponding amino acids by means of reverse-phase high-performance liquid chromatography (HPLC). Uptake of the reporter peptide was competitively inhibited by casein-derived peptides. The competition was only because of charged casein-derived peptides, including anionic peptides. The design of specific pure peptides made it possible to evidence for a positive (or negative) influence exerted by the positively (or negatively) charged side chain of the N-terminal amino acid on the competition. Conclusions: Charged casein-derived peptides impaired the oligopeptide transport function of L. lactis. Significance and Impact of the Study: These results demonstrate an inhibition of Opp when too many peptides are produced by the proteinase. Peptide transport by Opp therefore represents a bottleneck for increasing the growth rate of L. lactis in milk.

-Who determines the diversity of oligopeptide transport specificity in Lactococcus lactis? In this review our knowledge of oligopeptide transport by Opp in Lactococcus lactis is discussed. The specific oligopeptide transport system Opp is essential for growth of lactic acid bacteria in milk. This system belongs to the superfamily of ABC transporters containing a binding protein, generally described as the determinant of the specificity. The substrate specificity of this system, studied on one strain of lactococci, L. lactis MG1363, appears to be very different from that of other bacteria, especially Salmonella typhimurium. Relatively broad, this specificity is not fully representative of that of L. lactis species. In fact, each strain displays specific preferences for peptide transport. Despite binding protein OppA sequence varies to one strain to another, its variability cannot explain the observed diversity of substrate specificity. In L. lactis, OppA is essential for peptides transport but does not entirely determine the substrate specificity and its diversity among the species.Lactococcus lactis / oligopeptide transport / specificity / diversity Résumé -La présente revue se propose de synthétiser l'état de nos connaissances concernant le transport des oligopeptides par le système Opp chez Lactococcus lactis. Ce système est indispensable à la croissance des bactéries lactiques dans le lait. Il appartient à la famille des transporteurs ABC constitués d'une protéine affine : la protéine OppA, généralement décrite comme le déterminant de la spécificité du transporteur. La spécificité de substrat de ce système, définie sur une souche de lactocoque, L. lactis MG1363, apparaît très différente de celle d'autres bactéries modèles comme Salmonella typhimurium par exemple. Relativement large, elle n'est toutefois pas totalement représentative de l'espèce L. lactis. En effet, il existe une spécificité de transport des peptides propre à chaque souche. Bien que la séquence de la lipoprotéine OppA diffère d'une souche à l'autre, sa variabilité ne permet pas d'expliquer la diversité de la spécificité de substrat constatée. Chez L. lactis, OppA est indispensable au transport, mais elle ne détermine pas à elle seule la spécificité du transport des peptides et sa diversité au sein de l'espèce.Lactococcus lactis / transport oligopeptide / spécificité de substrat / diversité * Auteur correspondant : juillard@jouy.inra.fr 96 P. Charbonnel et al.