Pk. Dinsmore et al., A LEUCINE REPEAT MOTIF IN ABIA IS REQUIRED FOR RESISTANCE OF LACTOCOCCUS-LACTIS TO PHAGES REPRESENTING 3 SPECIES, Gene, 212(1), 1998, pp. 5-11
The abiA gene encodes an abortive bacteriophage infection mechanism th
at can protect Lactococcus species from infection by a variety of bact
eriophages including three unrelated phage species. Five heptad leucin
e repeats suggestive of a leucine zipper motif were identified between
residues 232 and 266 in the predicted amino acid sequence of the AbiA
protein. The biological role of residues in the repeats was investiga
ted by incorporating amino acid substitutions via site-directed mutage
nesis. Each mutant was tested for phage resistance against three phage
s, phi 31, sk1, and c2, belonging to species P335, 936, and c2, respec
tively. The five residues that comprise the heptad repeats were design
ated L234, L242, A249, L256, and L263. Three single conservative mutat
ions of leucine to valine in positions L235, L242, and L263 and a doub
le mutation of two leucines (L235 and L242) to valines did not affect
AbiA activity on any phages tested. Non-conservative single substituti
ons of charged amino acids for three of the leucines (L235, L242, and
L256) virtually eliminated AbiA activity on all phages tested. Substit
ution of the alanine residue in the third repeat (A249) with a charged
residue did not affect AbiA activity. Replacement of L242 with an ala
nine elimination phage resistance against phi 31, but partial resistan
ce to ski and c2 remained. Two single proline substitutions for leucin
es L242 and L263 virtually eliminated AbiA activity against all phages
, indicating that the predicted alpha-helical structure of this region
is important. Mutations in an adjacent region of basic amino acids ha
d various effects on phage resistance, suggesting that these basic res
idues are also important for AbiA activity. This directed mutagenesis
analysis of AbiA indicated that the leucine repeat structure is essent
ial for conferring phage resistance against three species of lactococc
al bacteriophages. (C) 1998 Elsevier Science B.V. All rights reserved.