Deregulation of the Arginine Deiminase ( arc ) Operon in Penicillin-Tolerant Mutants of Streptococcus gordonii

Abstract

Penicillin tolerance is an incompletely understood phenomenon that allows bacteria to resist drug-induced killing. Tolerance was studied with independent Streptococcus gordonii mutants generated by cyclic exposure to 500 times the MIC of penicillin. Parent cultures lost 4 to 5 log 10 CFU/ml of viable counts/24 h. In contrast, each of four independent mutant cultures lost <2 log 10 CFU/ml/24 h. The mutants had unchanged penicillinbinding 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 arc locus indicated that tolerance was not a direct consequence of arc alteration. On the other hand, genetic transformation of tolerance by Tol1 DNA always conferred arc deregulation. In nontolerant recipients, arc was repressed during exponential growth and up-regulated during postexponential growth. In tolerant transformants, arc was constitutively expressed. Tol1 DNA transformed tolerance at the same rate as transformation of a point mutation (10 ؊2 to 10 ؊3 ). The tolerance mutation mapped on a specific chromosomal fragment but was physically distant from arc. Importantly, arc deregulation was observed in most (6 of 10) of additional independent penicillin-tolerant mutants. Thus, although not exclusive, the association between arc deregulation and tolerance was not fortuitous. Since penicillin selection mimicked the antibiotic pressure operating in the clinical environment, arc deregulation might be an important correlate of naturally occurring tolerance and help in understanding the mechanism(s) underlying this clinically problematic phenotype.

Detection of Live and Antibiotic-Killed Bacteria by Quantitative Real-Time PCR of Specific Fragments of rRNA

“…A described streptomycin-resistant S. gordonii Challis strain and its penicillin-tolerant mutant Tol1 were used as model organisms (6,22). Streptococci were grown at 37°C without aeration either in brain heart infusion (Difco Laboratories, Detroit, MI) supplemented with 200 mg/liter of streptomycin (in order to respect the experimental conditions previously described for these isolates) (6,22) or on Columbia agar (Becton Dickinson Microbiology Systems, Cockeysville, MD) supplemented with 3% blood. Escherichia coli XL1-Blue cells were grown at 37°C in Luria-Bertani (LB) broth (Difco) or on LB agar (Difco), supplemented with 50 mg/liter of ampicillin (Sigma Chemicals).…”

“…This organism was used as a model because it is poorly lysed by penicillin and provides a well-described isogenic pair of kill-susceptible (parent) and kill-resistant (tolerant mutant Tol1) strains (6,22). Studies were performed with two mechanistically unrelated compounds, i.e., penicillin G and the quinolone levofloxacin.…”

Loss of Antibiotic Tolerance in Sod-Deficient Mutants Is Dependent on the Energy Source and Arginine Catabolism in Enterococci

“…Finally, the high-energy compound carbamoylphosphate is degraded into CO 2 and NH 3 with the concomitant production of an ATP by carbamate kinase encoded by arcC (15,16). However, inactivation of the arc locus in drug-tolerant strains of S. gordonii showed that increased tolerance was not a direct consequence of enhanced expression of the ADI pathway (14).…”

“…Furthermore, suppression of autolysis in Streptococcus pneumoniae and Staphylococcus aureus had no significant effect on killing by penicillin (6, 10-13). These data collectively indicated that a decrease in the autolytic activity could not fully explain tolerance to bactericidal antibiotics for all drug/pathogen combinations.Characterization of tolerant strains of Streptococcus gordonii that were obtained by cyclic exposure to penicillin at 500 times the MIC showed that two proteins belonging to the arc operon were upregulated in these derivatives (14). The arc operon encodes activities for arginine catabolism.…”

“…Characterization of tolerant strains of Streptococcus gordonii that were obtained by cyclic exposure to penicillin at 500 times the MIC showed that two proteins belonging to the arc operon were upregulated in these derivatives (14). The arc operon encodes activities for arginine catabolism.…”