[Mutational analysis of the conserved motif of the Arda anti-restriction protein encoded by self-transmissible IncI plasmid ColIb-p9].
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 plasmids 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 L130, L131, and V138 were substituted with negatively charged E; negatively charged D133, E136, and D143 substituted with hydrophobic V; and D127, D150, and D154 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.
Del'ver
E P
EP
Institute of Experimental Cardiology, Cardiology Research Center, Ministry of Health of the Russian Federation, Moscow, 121552 Russia.
Sobennikova
M V
MV
Belogurova
N G
NG
Nekrasov
S V
SV
Kolesnikova
M D
MD
Agafonova
O V
OV
Belogurov
A A
AA
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D015280:DNA Restriction-Modification Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D012097:Repressor Proteins
C096738:ArdA protein, E coli; D001426:Bacterial Proteins; D015280:DNA Restriction-Modification Enzymes; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D000409:Alanine