A 5-kb DNA fragment conferring a phage abortive infection phenotype (Abi+) has been cloned from Lactococcus lactis subsp. lactis IL416. The Abi+ determinant was subcloned on a 2-kb fragment which carried an Iso-ISSI element and an open reading frame of 753 bp designated ORFX. Deletion within ORFX entailed the loss of the Abi+ phenotype, establishing that ORFX is the structural abi416 gene. The expression of abi 416 was shown to be mediated by the Iso-ISSI element, which contains a sequence fitting the consensus sequence for gram-positive promoters.
Lactobacillus bulgaricus strains LT1 and LT4 previously isolated from a yogurt factory were shown to be lysogenic and inducible by mitomycin C or u.v. irradiation, induction being optimal at the beginning of exponential growth. Five indicator strains (four of L. lactis, one of L. bulgaricus) were found in which the two phage lysates propagated well in a liquid medium, but formed plaques on bacterial lawns less readily. Ca 2+ was required for the phage infection of the indicator strains to reach termination. Phage particles from each strain exhibited a comparable ultrastructural morphology under the electron microscope, having an isometric head, a triple collar and a noncontractile fibre-containing tail.
Lactobacillus bulgaricus
LT4(0448) is a lysogenic strain from which a temperate bacteriophage can be induced by mitomycin C or UV irradiation.
Lactobacillus lactis
CNRZ 326 is an indicator strain for the temperate phage 0448, but this strain lyses only in the presence of Ca
2+
ions. A resistant culture developed secondarily after phage lysis and grew normally in MRS broth but again lysed abruptly if Ca
2+
ions were added after two or three transfers. This behavior of the secondary culture and its subcultures is explained by a heterogeneous and fluctuating bacterial population, including clones identical to
L. lactis
326, which were sensitive to 0448 and which formed rough colonies, as does the indicator. The proportion of these clones increased in the course of transfers in MRS, explaining lysis when Ca
2+
was added. The population also included clones which formed smooth colonies (S clones). SI clones, which could not be induced by mitomycin C, were the major type in the initial culture, although they were sensitive to temperate phage 0448. The SI population then decreased and was gradually replaced by SII clones, inducible by mitomycin C and resistant to 0448. These SII clones were lysogenized clones, 326(0448), whose stability was confirmed by growth in the presence of an antiphage serum. When
L. bulgaricus
LT4(0448) was treated with mitomycin C, several cured LT4 clones were obtained that were related to the clones of the indicator
L. lactis
326; they formed rough colonies. They also became sensitive to lytic phages or temperate phages active against
L. lactis
326 and insensitive to lytic phages which lysed
L. bulgaricus
LT4(0448). This suggests that phage 0448 can lead to a lysogenic conversion of host strain LT4.
Résumé -Les phages sont reconnus depuis longtemps comme la principale cause de ralentissement de la croissance des lactocoques composant les levains de fromagerie. La sélection de souches naturelles ou mutantes particulièrement résistantes aux phages ne permettant pas un contrôle optimum des fabrications, une nouvelle approche a été envisagée après analyse des méca-nismes de résistance aux phages naturellement présents dans les souches. Il s'agit de construire des souches plus résistantes aux phages par accumulation de plusieurs mécanismes de résistance dont les effets s'additionnent. Une telle approche nécessite l'isolement et la caractérisation de méca-nismes variés de résistance aux phages. Nous résumons ici le clonage et la caractérisation des gènes codant pour 3 mécanismes d'infection abortive. Les cibles de ces 3 mécanismes ont été localisées sur l'ADN d'un phage. Deux d'entre elles ont été clonées et sont en cours de caractérisation. L'étude de l'interaction entre les gènes codant pour ces mécanismes de résistance et leur cible sur le phage devrait nous apporter les connaissances nécessaires pour un choix rationnel des méca-nismes à utiliser.
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