J . D IJ K ST ER H UI S, M . S AN D ER S, L .G .M . GO RR I S A ND E .J . S M ID . 1999.Interaction of Fusarium oxysporum and Paenibacillus polymyxa starts with polar attachment of bacteria to the fungal hyphae followed by the formation of a large cluster of non-motile cells embedded in an extracellular matrix in which the bacteria develop endospores. Enumeration of fungal viable counts showed that less than one of 36 000 colony-forming units survived in paired cultures for 71 h. Effective antagonism was not observed below pH 5 and was specific for the bacterial species. Development of F. oxysporum was inhibited in cell-free filtrates derived from cultures of P. polymyxa, but was much more strongly repressed in the presence of living bacteria. Furthermore, recovery of fungal growth started immediately after addition of antibiotics to paired cultures. Restoration of fungal growth was enhanced in filtrates that were supplemented with MgCl 2 , which suggests that anti-fungal compounds produced by the bacteria were counteracted by magnesium ions. In paired cultures, fungal counts remained very low, even in the presence of the magnesium salt.This study clearly showed that P. polymyxa antagonizes the plant pathogenic fungus F. oxysporum in liquid medium by means of an interaction process in which the presence of living bacteria is a prerequisite for continuous suppression of fungal growth.

The chlorated dipeptide L-alanyl-j-chloro-L-alanine (diACA) is very toxic for Lactococcus lactis. Spontaneous mutants resistant to the dipeptide were isolated from plates. The presence and activities of cell wall-associated proteinase, different peptidases in cell extracts, amino acid transport systems, and di-and oligopeptide transport systems were examined and compared in a diACA-resistant mutant and the wild type. Only the rates of di-and tripeptide transport were found to be significantly reduced in the diACA-resistant mutant ofL. lactis ML3. Since all other characteristics of this mutant were comparable to those of the wild type, the diACA-resistant mutant is most likely deficient in di-and tripeptide transport. Uptake of di-and tripeptides by L. lactis ML3 was found to be mainly mediated by one peptide transport system. The peptide transport-deficient mutant was found to be unable to grow on a chemically defined medium supplemented with casein as the sole nitrogen source, whereas growth could be restored by the addition of amino acids. These results indicate that peptide transport in L. lactis ML3 is an essential component in the process of casein utilization during growth in milk.In the dairy industry, lactococci (previously named group N lactic streptococci [19]) are commonly used as starter cultures in milk fermentations. These gram-positive bacteria are very fastidious and require exogenous sources of nucleotides, vitamins, and amino acids (16). Several amino acids are either stimulatory or essential for lactococcal growth (5, 16). The concentrations of free amino acids in milk are, however, too low to support optimal starter growth (22). For rapid acid production in milk fermentations, these organisms possess proteinases and a number of peptidases (see reference 22 for a recent review) which hydrolyze the milk protein casein and supply the cells with the required amino acids. Most strains of Lactococcus lactis can utilize amino acids and peptides with up to approximately four to six residues to satisfy their growth requirements (9,18,21,24).Hugenholtz et al. (5) showed that the rate of growth of L. lactis subsp. cremoris in milk is determined by the rate of casein hydrolysis and that casein limitation leads to a limitation of amino acids. These results raised the question of whether the casein-derived limiting amino acids were supplied as free amino acids or as small peptides.Different studies were undertaken to elucidate the actual pathway of casein utilization by lactococci. Proteolytic attack of bovine P-casein by the cell wall proteinases of L. lactis (12) and L. lactis subsp. cremoris (25, 26) can occur at seven cleavage positions at the C-terminal stretch of the milk protein. Proteolysis thus results in a mixture of peptide fragments which have to be further degraded by extracellular peptidases before they can pass through the cytoplasmic membrane. The information available about the extracellular breakdown of these peptide fragments is limited. It is still not clear how many extracellular pept...

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):

Alanyl-alpha-glutamate transport has been studied in Lactococcus lactis ML3 cells and in membrane vesicles fused with liposomes containing beefheart cytochrome c oxidase as a proton-motive-force-generating system. The uptake of Ala-Glu observed in de-energized cells can be stimulated 26-fold upon addition of lactose. No intracellular dipeptide pool could be detected in intact cells. In fused membranes, a 40-fold accumulation of Ala-Glu was observed in response to a proton motive force. Addition of ionophores and uncouplers resulted in a rapid efflux of the accumulated dipeptide, indicating that Ala-Glu accumulation is directly coupled to the proton motive force as a driving force. Ala-Glu uptake is an electrogenic process and the dipeptide is transported in symport with two protons. In both fused membranes and intact cells the same affinity constant (0.70 mM) for Ala-Glu uptake was found. Accumulated Ala-Glu is exchangeable with externally added alanyl-glutamate, glutamyl-glutamate, and leucyl-leucine, while no exchange occurred upon addition of the amino acid glutamate or alanine. These results indicate that the Ala-Glu transport system has a broad substrate specificity.

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