Relationship between utilization of proline and proline-containing peptides and growth of Lactococcus lactis

Abstract

Proline, which is the most abundant residue in I-casein, stimulates growth of Lactococcus lactis in a proline-requiring strain (Lactococcus lactis subsp. cremoris Wg2) and in a proline-prototrophic strain (Lactococcus lactis subsp. Iactis ML3). Both strains lack a proline-specific uptake system, and free proline can enter the cell only by passive diffusion across the cytoplasmic membrane. On the other hand, lactococci can actively take up proline-containing peptides via the lactococcal di-and tripeptide transport system, and these peptides are the major source of proline. Consequently, lactococcal growth on amino acid-based media is highly stimulated by the addition of proline-containing di-and tripeptides. Growth (Difco Laboratories, Detroit, Mich.) and stored at -80°C. For growth experiments, the cultures were transferred from milk cultures to complex broth medium (2) at a pH of 6.4 containing 0.5% (wt/vol) lactose as a carbon and energy source and incubated overnight at 28°C. The overnight cultures were subsequently transferred to chemically defined media containing 0.5% (wt/vol) lactose as a carbon and energy source. These cultures were used to inoculate the experimental cultures.The composition of the chemically defined medium (CDM) was as previously described (10). In all cases, glutamine and asparagine instead of glutamate and aspartate were used. Unless stated otherwise in the legends to figures, the amino acids were added to the medium at the following concentrations (mM):

Di-tripeptides and oligopeptides are taken up via distinct transport mechanisms in Lactococcus lactis

“…The high proline content of ,-casein (35 of 209 residues [21]) suggests that several essential and growth-stimulating case-in-derived amino acids will be supplied to the cells as proline-containing peptides. The finding that especially proline-containing dipeptides are high-affinity substrates for the lactococcal di-tripeptide transport system is in agreement with this notion (24).…”

BglR protein, which belongs to the BglG family of transcriptional antiterminators, is involved in beta-glucoside utilization in Lactococcus lactis

“…lactis IL1403 (11) and derivatives, E. coli TG1 (18) and MA152 (24), and B. subtilis MT119 (41) were grown as previously described (8). Growth on various carbohydrates in a chemically defined broth (28,29,38) procedure using CaCl2 (32) and the modified protocol (10) of Anagnostopoulos and Spizizen (5), respectively. L. lactis was transformed by an electroporation technique (20).…”

“…lactis IL1403 (11) and derivatives, E. coli TG1 (18) and MA152 (24), and B. subtilis MT119 (41) were grown as previously described (8). Growth on various carbohydrates in a chemically defined broth (28,29,38), supplemented with the appropriate sugar at 1% final concentration, was monitored. The medium was inoculated with 1% of a culture grown overnight and then washed twice.…”

Cloning, sequencing and characterization of the pepIP gene encoding a proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397

“…Therefore, proline-containing peptides resulting from casein degradation by a cellsurface proteinase have to be shortened before crossing the cell membrane because L. bdgaricns requires proline for its growth (Atlan et al, 1989). In L. bulgaricgs and L. helvetiens (data not shown), PepIP is located in the cell envelope and could initiate another catabolic pathway for such peptides ; its action combined with that of other cellwall peptidase (AP 11) would give rise to peptides short enough to be transferred ; free proline would enter the cell by passive diffusion (Smid & Konings, 1990). …”