Rho-specific Bacillus cereus ADP-ribosyltransferase C3cer cloning and characterization.

C3-like ADP-ribosyltransferases represent an expanding family of related exoenzymes, which are produced by Clostridia and various Staphylococcus aureus strains. Here we report on the cloning and biochemical characterization of an ADP-ribosyltransferase from Bacillus cereus strain 2339. The transferase encompasses 219 amino acids; it has a predicted mass of 25.2 kDa and a theoretical isoelectric point of 9.3. To indicate the relationship to the family of C3-like ADP-ribosyltransferases, we termed the enzyme C3cer. The amino acid sequence of C3cer is 30 to 40% identical to other C3-like exoenzymes. By site-directed mutagenesis, Arg(59), Arg(97), Tyr(151), Arg(155), Thr(178), Tyr(180), Gln(183), and Glu(185) of recombinant C3cer were identified as pivotal residues of enzyme activity and/or protein substrate recognition. Precipitation experiments with immobilized RhoA revealed that C3cerTyr(180), which is located in the so-called "ADP-ribosylating toxin turn-turn" (ARTT) motif, plays a major role in the recognition of RhoA. Like other C3-like exoenzymes, C3cer ADP-ribosylates preferentially RhoA and RhoB and to a much lesser extent RhoC. Because the cellular accessibility of recombinant C3cer is low, a fusion toxin (C2IN-C3cer), consisting of the N-terminal 225 amino acid residues of the enzyme component of C2 toxin from Clostridium botulinum and C3cer was used to study the cytotoxic effects of the transferase. This fusion toxin caused rounding up of Vero cells comparable to the effects of Rho-inactivating toxins.