A leucine repeat motif in AbiA is required for resistance of Lactococcus lactis to phages representing three species.
The abiA gene encodes an abortive bacteriophage infection mechanism that can protect Lactococcus species from infection by a variety of bacteriophages including three unrelated phage species. Five heptad leucine 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 investigated by incorporating amino acid substitutions via site-directed mutagenesis. Each mutant was tested for phage resistance against three phages, phi 31, sk1, and c2, belonging to species P335, 936, and c2, respectively. The five residues that comprise the heptad repeats were designated L234, L242, A249, L256, and L263. Three single conservative mutations of leucine to valine in positions L235, L242, and L263 and a double mutation of two leucines (L235 and L242) to valines did not affect AbiA activity on any phages tested. Non-conservative single substitutions of charged amino acids for three of the leucines (L235, L242, and L256) virtually eliminated AbiA activity on all phages tested. Substitution of the alanine residue in the third repeat (A249) with a charged residue did not affect AbiA activity. Replacement of L242 with an alanine elimination phage resistance against phi 31, but partial resistance to sk1 and c2 remained. Two single proline substitutions for leucines 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 had various effects on phage resistance, suggesting that these basic residues are also important for AbiA activity. This directed mutagenesis analysis of AbiA indicated that the leucine repeat structure is essential for conferring phage resistance against three species of lactococcal bacteriophages.
Dinsmore
P K
PK
Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh 27695, USA.
O'Sullivan
D J
DJ
Klaenhammer
T R
TR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D001435:Bacteriophages / Q000472:pathogenicity*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D003611:Dairy Products / Q000382:microbiology; D005798:Genes, Bacterial; D013294:Lactococcus lactis / Q000235:genetics* / Q000821:virology*; D007930:Leucine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D013045:Species Specificity; D014774:Virulence
D001426:Bacterial Proteins; D017931:DNA Primers; D007930:Leucine