A multifunction ABC transporter (Opt) contributes to diversity of peptide uptake specificity within the genus Lactococcus

Abstract

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.

FIG. 1.

Growth of L. lactis SK11 and its Opp⁻ derivative SKM6 in the presence of peptide as a source of essential amino acids. Growth rate was compared to that obtained in CDM containing all the amino acids in free form.

FIG. 2.

Variability of oligopeptide transport system expression by L. lactis. Immunodetection of OBP proteins OppA (A) or OptA (B) or cDNA amplification of the region optA-optB (C) or optS (D) in L. lactis MG1363 (lane 1), IL-1403 (lane 2), SK11 (lane 3), SKM6 (lane 4), WG2 (lane 5), MLS (lane 6), and MLA (lane 7). The molecular mass marker used in cDNA fragment electrophoresis was the 1-kb DNA ladder (Invitrogen).

FIG.3.

Sequence comparison of OBPs from gram-positive bacteria. OptA, OptS, and OppA sequences of L. lactis IL-1403 were obtained from the corresponding sequenced genome (4). The sequences of OppA2 from S. uberis 0140J, OppA of B. subtilis 168, OppA of L. lactis Wg2, OppA1 of L. delbrueckii subsp. bulgaricus B14, AmiA1 of S. thermophilus, and AmiA of S. pneumoniae R800 were obtained from databases by their respective accession numbers (GenBank accession no. AY256913, P24141, AY189901, AAK72116, AAL68705.1, and AE007579, respectively). Identity of 75% and similarity of 85% of the amino acids are indicated by boxes and by the grey background, respectively.

FIG. 4.

Growth of L. lactis ILM521 synthesizing the cell surface proteinase PrtP in milk (▪) and its opt derivatives MLA521 (OptA⁻; •) and MLS521 (OptS⁻; ○). Milk was supplemented with 0.5% (wt/vol) glucose. The assay was repeated twice.

FIG. 5.

Neighbor-joining phylogenetic tree of OBP sequences. The tree was inferred by using an expansion of CLUSTALW by Infobiogen (http://www.infobiogen.fr). Reading from top to bottom of the tree, the following sequences were obtained from databases by their respective GenBank accession numbers (in parentheses): AmiA1, AmiA2, and AmiA3 of S. thermophilus ST18 (AAL68705, AAL68706, and AAL68707), AmiA1 of S. pneumoniae R800 (AE007579), HppA of S. gordonii DL1 (AAB46614.1), AliB of S. pneumoniae R800 (Q51933), OppA of S. mutans UA159 (AAN58024.1), OppA2 from S. uberis 0140J (AY256913), OptS and OptA from the sequenced genome of L. lactis IL-1403 (4), OppA of E. coli K-12 (P23843), OppA of B. subtilis 168 (P24141), AppA of B. subtilis JH12795 (P42061), OppA2 and OppA1 of L. delbrueckii subsp. bulgaricus B14 (AAK72116), OppA of L. lactis Wg2 (AY189901), and OppA of L. lactis IL-1403 (4).