Aims: The present work aimed to define a minimal chemically‐defined medium which could sustain the growth of most (if not all) strains of Streptococcus thermophilus. Methods and Results: A minimal medium containing 20 components, including one carbohydrate source, six amino acids, two metallic ions, six vitamins and urea allowed for growth of 13 out of 15 Strep. thermophilus strains. Growth of the two last strains required the presence of additional amino acids, the number of which depended on the strain. Growth rates of the strains in the minimal medium ranged from 0·38 to 0·64 h–1, and final populations were about 108 cfu ml–1. Conclusions: Streptococcus thermophilus appears much less demanding than other lactic acid bacteria. Significance and Impact of the Study: The definition of such a growth medium will be very useful for metabolic flux studies as well as peptide transport studies.

The peptides released from ␤-casein by the action of P I -type proteinase (PrtP) from Lactococcus lactis subsp. cremoris Wg2 have been identified by on-line coupling of liquid chromatography to mass spectrometry. After 24 h of incubation of ␤-casein with purified PrtP, a stable mixture of peptides was obtained. The trifluoroacetic acid-soluble peptides of this ␤-casein hydrolysate were fractionated by high-performance liquid chromatography and introduced into the liquid chromatography-ion spray mass spectrometry interface. Multiply charged ions were generated from trifluoroacetic acid-soluble peptides under low nozzle voltage conditions, yielding the MH ؉ mass of each eluted peptide. All peptides corresponding to each of the MH ؉ calculated masses were determined. In those cases in which different peptides were possible, further identification was achieved by collision-induced dissociation under higher nozzle voltage conditions. Hydrolysis of ␤-casein by PrtP was observed to proceed much further than reported previously. More than 40% of the peptide bonds are cleaved by PrtP, resulting in the formation of more than 100 different oligopeptides. With the exception of Phe, significant release of amino acids or di-and tripeptides could not be observed. Interestingly, one-fifth of the identified oligopeptides are small enough to be taken up by the oligopeptide transport system. Uptake of these peptides could supply L. lactis with all amino acids, including the essential ones, indicating that growth of L. lactis might be possible on peptides released from ␤-casein by proteinase only.Lactococci have very limited capacities of synthesizing amino acids and therefore must utilize exogenous nitrogen sources for optimal growth. The amino acid requirement is strain dependent, but Glu or Gln, Ile, Leu, His, Met, and Val are essential for the growth of most Lactococcus lactis strains (6). The addition of several other amino acids was found to be growth stimulatory (34,25). The concentrations of essential amino acids in milk are very low, especially those of Ile and Leu (less than 1 mg/liter) (27). Moreover, the concentrations of other free amino acids are too low to explain the growth of L. lactis to the cell densities normally reached in coagulated milk (27,44). Thus, for optimal growth in milk, lactococci depend on the utilization of milk proteins, such as caseins. Casein hydrolysis by lactococci is mediated by a complex proteolytic system which includes a cell envelope-located proteinase (P I -or P III -type proteinase [PrtP]) and several peptidases (for recent reviews, see references 31, 32, and 42).According to proposed models, PrtP is involved in the first step of casein degradation (32, 39). The action of purified PrtP on ␤-casein has been studied extensively (28,29,33,46,47). After separation of the proteolytic products by reverse-phase high-performance liquid chromatography (HPLC), the different peptides are collected, purified when needed, and identified by biochemical methods (i.e., amino acid composition, determina...

In the proteolytic pathway of Lactococcus lactis, milk proteins (caseins) are hydrolyzed extracellularly to oligopeptides by the proteinase (PrtP). The fate of these peptides, i.e. extracellular hydrolysis followed by amino acid uptake or transport followed by intracellular hydrolysis, has been addressed. Mutants have been constructed that lack a functional di-tripeptide transport system (DtpT) and/or oligopeptide transport system (Opp) but do express the P1-type proteinase (specific for hydrolysis of beta- and to a lesser extent kappa-casein). The wild type strain and the DtpT- mutant accumulate all beta-casein-derived amino acids in the presence of beta-casein as protein substrate and glucose as a source of metabolic energy. The amino acids are not accumulated significantly inside the cells by the Opp- and DtpT- Opp- mutants. When cells are incubated with a mixture of amino acids mimicking the composition of beta-casein, the amino acids are taken up to the same extent in all four strains. Analysis of the extracellular peptide fraction, formed by the action of PrtP on beta-casein, indicates that distinct peptides disappear only when the cells express an active Opp system. These and other experiments indicate that (i) oligopeptide transport is essential for the accumulation of all beta-casein-derived amino acids, (ii) the activity of the Opp system is sufficiently high to support high growth rates on beta-casein provided leucine and histidine are present as free amino acids, and (iii) extracellular peptidase activity is not present in L. lactis.

The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used.