S U M M A R YWhen staphylococci, resistant to 4 pg. tetracycline/ml., were grown in nutrient media at subinhibitory levels of the drug, phenotypical resistance increased until the cocci grew with 160 pg. tetracyclinelml. Resistance increased most rapidly at the highest concentration of tetracycline which did not significantly inhibit growth. Increase in resistance was also obtained by pre-incubation with p-apo-5-oxy-tetracycline. Increase in resistance could be prevented by chloramphenicol and actinomycin D, but not by nalidixic acid. When a highly resistant culture was transferred to tetracycline-free medium, phenotypical resistance decreased gradually ; after four transfers on nutrient agar it returned entirely to the original level.
I N T R O D U C T I O NWilliams (1967) has drawn attention to the resistance to mercury salts (Moore, I 960) of tetracycline-resistant strains of Staphylococcus aureus. Resistance to tetracycline was in general correlated with resistance to mercury; in mercury-sensitive strains, however, high-level resistance to tetracycline could be obtained by ' training ', i.e. growth in peptone water containing 3 pg. antibiotic/ml.
METHODSBacterial strains. The strain studied, Staphjdococcus aureus I I I (Sompolinsky, Yiflah & Aboud, 1568), was susceptible to mercuiy salts (Moore, 1960) and resistant to tetracycline (Tc) by virtue of a plasmid that could be eliminated by growth at 44" (May, Houghton & Perret, 1964). The eliminated susceptible strain grew on nutrient agar containing 0.2 pg. Tc/ml. ; on agar containing 0.4 pg. Tc/ml., colonies did not develop from an inoculum of IO* staphylococci. With the uneliminated culture, identical colony counts were obtained on plain agar and on agar containing 4-0 pg. Tc/ml. At concentrations of up to 160pg. Tc/ml., some colonies developed but growth did not occur on nutrient agar containing 320pg. Tc/ml. Three types of Iesistant cultures have been used: (a) a culture transferred daily on plain nutrient agar; only 5 to 10% of the viable cocci from this culture produced colonies on agar containing 10 pg. Tc/ml. ; (b) a highly resistant culture subcultured daily on nutrient agar containing I 60 pg. Tc/ml.; and (c) cultures with intermediate levels of resistance obtained by growth of type (a) culture for a short period in nutrient broth containing tetracycline at different concentrations.
Variants of chloramphenicol acetyltransferase from a variety of bacterial species have been isolated and purified to homogeneity. They constitute a heterogeneous group of proteins as judged by analytical affinity and hydrophobic (‘detergent’) chromatography, native and sodium dodecyl sulfate electrophoresis, sensitivity to sulfhydryl specific reagents, steady state kinetic analysis, and reaction with antisera.
The most striking observation is that three variants of chloramphenicol acetyltransferase (R factor type III, Streptomyces acrimycini, and Agrobacterium tumefaciens) possess an apparent subunit molecular weight (24500) which is significantly greater than that of all other variants examined (22500). The three atypical variants are not identical since they show marked differences in a number of important parameters.
Although the fundamental mechanism of catalysis may prove to be identical for all chloramphenicol acetyltransferase variants, there is a wide range of sensitivity to thiol‐directed inhibitors among the enzymes studied.
Amino acid sequence analysis of the N‐termini of selected variants suggests that the qualitative differences among chloramphenicol acetyltransferase variants is a reflection of structural heterogeneity which is most marked in comparisons between variants from Gram‐positive and Gram‐negative species.
1. The commonly observed type I variant of chloramphenicol acetyltransferase specified by R factors is a tetrameric enzyme and contains four half-cysteine residues per identical subunit.All four thiols are reactive when the protein has been reduced in the presence of 6 M guanidine-HC1.
2.Iodoacetamide is an effective inhibitor of native chloramphenicol acetyltransferase. The incubation of iodo[14C]acetamide with enzyme leads to amido-carboxymethylation. Chloramphenicol is effective in preventing both inactivation and chemical modification, whereas acetyl-CoA fails to protect against either effect. Evidence is presented in support of the preferential reactivity of a single cysteine residue (SI) towards iodoacetamide. Iodoacetate is not an effective inhibitor and fails to react with the native enzyme under conditions which permit inactivation and chemical modification by iodoacetamide.3. The chromogenic disulfides 2,2'-dithio-bis(pyridine) and 4,4'-dithio-bis(pyridine) are more effective at low concentrations than iodoacetamide as inhibitors of chloramphenicol acetyltransferase. Inhibition and covalent modification of chloramphenicol acetyltransferase by 2,2'-dithiobis(pyridine) can be prevented by chloramphenicol but not by acetyl-CoA. The same half-cq steinecontaining sequence (S1), adduced to be the site of alkylation by iodoacetamide, appears to be uniquely susceptible to mixed disulfide formation with 2,2'-dithio-bis(pyridine). Cyanylation of the 2-thiopyridyl-inhibited enzyme does not lead to reactivation.4. Chloramphenicol acetyltransferase is also susceptible to inhibition by N-ethylmaleimide, pmercuribenzoate, and 1 -fluoro-2,4-dinitrobenzene, and protection is afforded in each case by chloramphenicol. Dinitrophenylation of one or more cysteine residues is inferred by the reactivation observed by incubation of the inactivated enzyme with reduced thiols.5. The reaction of i~do['~C]acetarnide with native chloramphenicol acetyltransferase followed by proteolytic digestion yields radioactive peptides corresponding to only three of the four known halfcysteine-containing sequences. The results of competition and protection experiments suggest that the S1 sequence is the preferential site of attack by iodoacetamide or 2,2'-dithio-bis(pyridine) and that it is sufficiently close to the chloramphenicol binding site to be protected by that substrate. A thiolcontaining sequence designated S3 has been judged to be implicated in the binding of acetyl-CoA on the basis of (a) protection against iodoacetamide labelling and inactivation by that substrate, and (b) probable covalent and selective modification of the S3 thiol by bromoacetyl-CoA.6. Low but reproducible rates of enzyme inactivation occur when chloramphenicol acetyltransferase is incubated in the presence of a number of CoA-related compounds. The inactivation process is not related to the low rates of hydrolysis of acyl-CoA derivatives, which have been noted in the absence of chloramphenicol. There is no evidence to suggest a sequential ('ping-pong') reaction me...
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