Lait 73 (1993) 199-205
DOI: 10.1051/lait:1993218

Amélioration de la résistance des lactocoques aux bactériophages

E. Bidnenko^a, R. Valyasevi^a, P.J. Cluzel^a, R. Parreira^a, A. Hillier^{a, b}, M. Gautier^c, J. Anba^a, S.D. Ehrlich^a and M.C. Chopin<sup>a

a</sup>  Laboratoire de génétique microbienne, INRA, 78352 Jouy-en-Josas
^b  Adresse permanente : CSIRO, Dairy Research Laboratory, PO Box 20, Highett, Victoria 3190, Australie
^c  Laboratoire de recherches de technologie laitière, INRA, 35032 Rennes, France

Abstract - Improvement of phage resistance in Lactococcus
Improvement of phage resistance in Lactococcus. The need to improve phage resistance in lactococcal strains used for cheesemaking has long been recognised. Recent molecular and genetic studies of these microorganisms have given insight into phage defense mechanisms and opened new possibilities for the improvement of lactococcal phage resistance. Naturally occur-ring lactococci with a high degree of phage resistance have been shown to possess several types of phage resistance mechanisms including adsorption interference, restriction and modification, and abortive infection. It has been suggested that cheesemaking strains might be improved by introducing several defense mechanisms, acting on a broad range of phages at different steps of their life cycle, into these strains. Such an approach relies on the availability of a number of highly efficient, well-characterized phage resistance mechanisms and on the development of efficient food grade transfer procedures in industrial strains. In this paper, we consider only the characterization of phage abortive infection mechanisms (Abi). In the presence of an Abi mechanism, the efficiency of phage adsorption remains the same but phages multiply only in a fraction of the infected cells. In addition, burst size is decreased. As a result, phage number does not increase and the culture grows normally. The Abi mechanisms are therefore of particular interest for the improvement of lactococcal phage résistance. In order to obtain more insight in the mode(s) of action of Abi mechanisms, we cloned 3 different Abi-encoding DNA fragments. These 3 determinants were compared to those already published or currently under characterization in other laboratories. Among 7 genes compared, 6 are different including the 3 we have cloned. These results indicate that Abi mechanisms are diverse and should have different modes of action. Two of the cloned determinants were subcloned and sequenced. In both cases, an iso-ISS1 element was present upstream of the gene coding for the Abi+ phenotype. The expression of both abi genes was directed by a promoter present in this IS element. Therefore, in addition to a contribution to lactococcal gene mobility, ISS1 is able to participate in gene expression. In order to understand how the abi gene products interfere with phage development, we have compared the small isometric-headed phage blL66 to one of its spontaneous mutants able to overcome abi-105. The mutation was localised on a 3-kb DNA fragment from the phage. In presence of abi-105, the 3 kb fragment carrying the mutation allows multiplication of several unrelated phages sensitive to abi-105. The gene involved in the multiplication of unrelated phages was localized on the 3-kb DNA fragment and sequenced. The protein it specifies shares no homology with proteins from the data banks. Studies are currently in progress to determine how the abi gene product interferes with the phage genes.

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 mécanismes 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 mécanismes 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 mécanismes à utiliser.

Key words: Lactococcus / bacteriophage / resistance / abortive infection

Mots clés : Lactococcus / bactériophage / résistance / infection abortive