Characterization of a rifampin-inactivating glycosyltransferase from a screen of environmental actinomycetes

Abstract

Identifying and understanding the collection of all antibiotic resistance determinants presented in the global microbiota, the antibiotic resistome, provides insight into the evolution of antibiotic resistance and critical information for the development of future antimicrobials. The rifamycins are broad-spectrum antibiotics that target bacterial transcription by inhibition of RNA polymerase. Although mutational alteration of the drug target is the predominant mechanism of resistance to this family of antibiotics in the clinic, a number of diverse inactivation mechanisms have also been reported. In this report, we investigate a subset of environmental rifampin-resistant actinomycete isolates and identify a diverse collection of rifampin inactivation mechanisms. We describe a single isolate, WAC1438, capable of inactivating rifampin by glycosylation. A draft genome sequence of WAC1438 (most closely related to Streptomyces speibonae, according to a 16S rRNA gene comparison) was assembled, and the associated rifampin glycosyltransferase open reading frame, rgt1438, was identified. The role of rgt1438 in rifampin resistance was confirmed by its disruption in the bacterial chromosome, resulting in a loss of antibiotic inactivation and a 4-fold decrease in MIC. Interestingly, examination of the RNA polymerase β-subunit sequence of WAC1438 suggests that it harbors a resistant target and thus possesses dual mechanisms of rifamycin resistance. Using an in vitro assay with purified enzyme, Rgt1438 could inactivate a variety of rifamycin antibiotics with comparable steady-state kinetics constants. Our results identify rgt1438 as a rifampin resistance determinant from WAC1438 capable of inactivating an assortment of rifamycins, adding a new element to the rifampin resistome.

Fig 1

(A) Structures of various rifamycins. (B) The glycosylation of rifampin catalyzed by Rgt1438, yielding 23-O-gluc-rifampin.

Fig 2

Phylogenetic analysis of Rgt1438 with proteins highly homologous to Rgt and natural product glycosyltransferases. Protein sequences were aligned using MUSCLE (14) and a Bayesian phylogenetic analysis was conducted using MrBayes (36) as described in Materials and Methods. Complete lists of proteins (including accession numbers) are located in Table S4 in the supplemental material.

Fig 3

Genetic organization of rgt1438 and surrounding open reading frames in WAC1438.

Fig 4

The rgt1438 gene product is responsible for the glycosylative inactivation of rifampin. (A) WAC1438 strains were grown for 3 days at 30°C in liquid SIM, at which point rifampin was added at a concentration of 20 μg/ml, representing time 0 h. Cultures were grown for an additional 24 h, and supernatants were sampled. Supernatants (10 μl) were applied on filter paper disks overlaid on a lawn of B. subtilis 168 and incubated overnight. A control sample included SIM supplemented with rifampin (20 μg/ml). Thiostrepton (Thio [inducer]) was added to achieve a final concentration of 1 μg/ml. (B) Supernatant samples mentioned above were diluted with equal volumes of methanol, centrifuged, and analyzed by HPLC. 1, control (SIM with rifampin); 2, WAC1438; 3, PTS1; 4, PTS2 plus thiostrepton.