The Tetracycline Destructases: A Novel Family of Tetracycline-Inactivating Enzymes

Abstract

Enzymes capable of inactivating tetracycline are paradoxically rare compared with enzymes that inactivate other natural-product antibiotics. We describe a family of flavoenzymes, previously unrecognizable as resistance genes, which are capable of degrading tetracycline antibiotics. From soil functional metagenomic selections, we discovered nine genes that confer high-level tetracycline resistance by enzymatic inactivation. We also demonstrate that a tenth enzyme, an uncharacterized homolog in the human pathogen Legionella longbeachae, similarly inactivates tetracycline. These enzymes catalyze the oxidation of tetracyclines in vitro both by known mechanisms and via previously undescribed activity. Tetracycline-inactivation genes were identified in diverse soil types, encompass substantial sequence diversity, and are adjacent to genes implicated in horizontal gene transfer. Because tetracycline inactivation is scarcely observed in hospitals, these enzymes may fill an empty niche in pathogenic organisms, and should therefore be monitored for their dissemination potential into the clinic.

Figure 1

Tetracycline-inactivating proteins. (A) Ten proteins derived from soil metagenomes and four tetracycline-inactivating proteins from NCBI. Numbers following NCBI sequences indicate GenInfo identifiers. Tet(56) was cloned from Legionella longbeachae. Asterisks denote nodes with Shimodaira-Hasegawa like branch supports >0.95 and circles denote nodes with support >0.7. Blue labels indicate proteins with 86% amino acid identity to one another. The scale bar represents the number of substitutions per site. (B) The minimum inhibitory concentrations (MICs) of E. coli heterologously expressing the indicated proteins. (C) Absolute tetracycline levels in media conditioned by E. coli strains expressing the designated proteins. “Theoretical Max” indicates the initial tetracycline concentration in the media, prior to inoculation.

Figure 2

E. coli transformants expressing either a tetracycline-resistant transporter or the indicated tetracycline-inactivating protein were grown in LB at 37°C for 4 days, protected from light. The same cultures expressing the tetracycline-resistant transporter are used across each image. Tetracycline was added at 100ug/ml except for Tet(55); 32ug/ml tetracycline was added to this sample due to a lower degree of tetracycline resistance conferred by this enzyme.

Figure 3

UV-visible spectrum of enzymatic tetracycline degradation. (A-L) Each panel shows the degradation of tetracycline over the course of three hours, in a reaction containing the indicated purified enzyme (or control), tetracycline, and an NADPH regeneration system. Absorbance scans were taken at one minute intervals. The rainbow pattern depicts a spectral change over time; absorbance at 360nm or 400nm always decreased with time. ORF; open reading frame. See also figure S7.

Figure 4

Tetracycline degradation is catalyzed by diverse flavoenzymes. (A-E) Reverse phase HPLC separation of tetracycline and enzymatically-catalyzed degradation products; absorbance at 260nm is shown. (F-J) The relative ion counts attributable to tetracycline (m/z for [M+H]⁺ equals 445 in positive ion mode) and products with m/z values of 461 and 387; data generated from the same reactions depicted in (A-E). The replacement of tetracycline with a product of +16 Da is consistent with monooxidation of the antibiotic and the mechanism through which Tet(X) catalyzes its degradation (19). A putative structure for the product with m/z = 387 is proposed in figures 6 and S6. Flavoenzymes from soil catalyze tetracycline degradation in manners both consistent with (e.g. B, G) and alternative to (e.g. A, F) Tet(X)-mediated catalysis (D, I). Data from experiments using all purified enzymes, oxytetracycline as substrate, and measurements of absorbance at 363nm are shown in figures S2, S3, S4, and S7.

Figure 5

Tet(X), but no other flavoenzyme, oxidizes anhydrotetracycline. (A-E) Representative UV-visible spectra; each panel shows absorbance spectra taken every 30 minutes throughout a 3.5 hour reaction with anhydrotetracycline and the indicated enzyme. The legend in (E) applies to (A-E); Tet(X) was the only flavoenzyme to show activity towards anhydrotetracycline. (F-J) The relative ion counts attributable to anhydrotetracycline (m/z for [M+H]⁺ equals 427 in positive ion mode) and a product with +16 Da, consistent with monoxidation of the substrate. (K-M) Representative LC-MS spectra of the indicated ions in from panel (I), measured at 3.5 hours as indicated in red. TIC; total ion count. EIC; extracted ion count.

Figure 6

The enzymatic conversions discussed within this manuscript. (A) Monooxygenation of tetracycline to compound 1, as described in ref. (19). (B) The proposed tetracycline oxidation products, compounds 2a and 2b, with an m/z value of 387. (C) Monooxygenation of anhydrotetracycline, depicted as is described for Tet(X)-catalyzed oxidation of tetracyclines in ref. (19).