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ABSTRACTPenicillin tolerance is an incompletely understood phenomenon that allows bacteria to resist drug-induced killing. Tolerance was studied with independent Streptococcus gordoniimutants generated by cyclic exposure to 500 times the MIC of penicillin. Parent cultures lost 4 to 5 log10CFU/ml of viable counts/24 h. In contrast, each of four independent mutant cultures lost ≤2 log10CFU/ml/24 h. The mutants had unchanged penicillin-binding proteins but contained increased amounts of two proteins with respective masses of ca. 50 and 45 kDa. One mutant (Tol1) was further characterized. The two proteins showing increased levels were homologous to the arginine deiminase and ornithine carbamoyl transferase of other gram-positive bacteria and were encoded by an operon that was >80% similar to the arginine-deiminase (arc) operon of these organisms. Partial nucleotide sequencing and insertion inactivation of the S. gordonii arclocus indicated that tolerance was not a direct consequence of arcalteration. On the other hand, genetic transformation of tolerance by Tol1 DNA always conferredarcderegulation. In nontolerant recipients,arcwas repressed during exponential growth and up-regulated during postexponential growth. In tolerant transformants,arcwas constitutively expressed. Tol1 DNA transformed tolerance at the same rate as transformation of a point mutation (10−2to 10−3). The tolerance mutation mapped on a specific chromosomal fragment but was physically distant fromarc. Importantly, arcderegulation was observed in most (6 of 10) of additional independent penicillin-tolerant mutants. Thus, although not exclusive, the association betweenarcderegulation and tolerance was not fortuitous. Since penicillin selection mimicked the antibiotic pressure operating in the clinical environment, arcderegulation might be an important correlate of naturally occurring tolerance and help in understanding the mechanism(s) underlying this clinically problematic phenotype.