Mutational analysis of the conserved motif of the ArdA antirestriction protein encoded by self-transmissible IncI plasmid ColIb-P9

作者：Delver EP^*; Sobennikova MV; Belogurova NG; Nekrasov SV; Kolesnikova MD; Agafonova OV; Belogurov AA

来源：Molecular Biology, 2002, 36(6): 864-868.

DOI：10.1023/A:1021646328954

• DNA restriction
• antirestriction proteins
• self-transmissible plasmids

A study was made of the functional role of the ArdA antirestriction motif (130-LLADVPETVALYFD- 143) conserved among all known Ard (alleviation of restriction of DNA) proteins, which are encoded by self-transmissible plastnids and specifically inhibit type I restriction-modification systems. Conserved residues of the motif were individually changed, and the resulting mutants tested for in vivo activity. Hydrophobic L 130, L131, and V 138 were substituted with negatively charged E; negatively charged D133, E136, and D143 substituted with hydrophobic V; and D 127, D 150, and D 154 neighboring the antirestriction motif substituted with V. Four substitutions (L130E, L131E, V138E, and D143V) substantially (25-1000 times) reduced the ArdA activity. The other substitutions within or beyond the motif had no appreciable effect. Substitutions L130A and L131A each reduced the ArdA activity 10- to 20-fold, indicating that high hydrophobicity of L130 and L131 is important for the ArdA function. Thus, the antirestriction role of ArdA is indeed due to its conserved motif.

• 出版日期2002-12