Identification of the biosynthetic gene cluster and an additional gene for resistance to the antituberculosis drug capreomycin

Abstract

Capreomycin (CMN) belongs to the tuberactinomycin family of nonribosomal peptide antibiotics that are essential components of the drug arsenal for the treatment of multidrug-resistant tuberculosis. Members of this antibiotic family target the ribosomes of sensitive bacteria and disrupt the function of both subunits of the ribosome. Resistance to these antibiotics in Mycobacterium species arises due to mutations in the genes coding for the 16S or 23S rRNA but can also arise due to mutations in a gene coding for an rRNA-modifying enzyme, TlyA. While Mycobacterium species develop resistance due to alterations in the drug target, it has been proposed that the CMN-producing bacterium, Saccharothrix mutabilis subsp. capreolus, uses CMN modification as a mechanism for resistance rather than ribosome modification. To better understand CMN biosynthesis and resistance in S. mutabilis subsp. capreolus, we focused on the identification of the CMN biosynthetic gene cluster in this bacterium. Here, we describe the cloning and sequence analysis of the CMN biosynthetic gene cluster from S. mutabilis subsp. capreolus ATCC 23892. We provide evidence for the heterologous production of CMN in the genetically tractable bacterium Streptomyces lividans 1326. Finally, we present data supporting the existence of an additional CMN resistance gene. Initial work suggests that this resistance gene codes for an rRNA-modifying enzyme that results in the formation of CMN-resistant ribosomes that are also resistant to the aminoglycoside antibiotic kanamycin. Thus, S. mutabilis subsp. capreolus may also use ribosome modification as a mechanism for CMN resistance.

FIG. 1.

Chemical structures of the four derivatives that make up CMN (left) and the structurally related tuberactinomycin antibiotic VIO (right). Numbering of residues within structures corresponds to residue numbers mentioned in the text. R₁, residue 1; R₂, residue 2.

FIG. 2.

Schematic of the 33 S. mutabilis subsp. capreolus ORFs contained on the pCMN-P4C8RF cosmid. The proposed CMN biosynthetic gene cluster consists of ORFs highlighted in gray and black. Those ORFs coding for proteins with significant sequence identity to proteins encoded by the VIO biosynthetic gene cluster are shown in gray. The newly identified CMN resistance gene is shown in black. The remaining ORFs flanking the proposed CMN biosynthetic gene cluster are shown in white.

FIG. 3.

HPLC traces of authentic CMN (A), metabolites purified from strain EAF1001 (S. lividans 1326 containing the integrated pCMN-P4C8RF-436 cosmid) (B), and metabolites purified from EAF1003 (S. lividans 1326 containing the integrated pOJ436 cosmid) (C). Metabolite elution was monitored at 268 nm. Arrows above peaks in trace A identify the peaks associated with the coelution of CMN IA and IB and the coelution of CMN IIA and IIB. Twenty milligrams of authentic CMN was injected. For EAF1001 and EAF1003 samples, 100 ml of culture was used in the purification. The purified compounds were resuspended in 500 μl of H₂O. A portion of these samples was diluted 1:10 in H₂O, and 20 μl of these diluted samples was injected into the HPLC column.

FIG. 4.

Schematic for the proposed NRPS for the formation of the cyclic pentapeptide core of CMN (CmnF, CmnA, CmnF, CmnJ, and CmnG), followed by downstream carbamoylation by CmnL and the acylation of CMN IIA and IIB by the combined functions of CmnM and CmnO. The individual domains of the NRPS are noted as circles with the appropriate abbreviation to denote function. Abbreviations are as follows: A, adenylation domain; PCP, peptidyl carrier protein domain; C, condensation domain; X, domain of unknown function; and C/, modified condensation domain. The arrows above the NRPS components depict the direction of pentapeptide synthesis on the NRPS. The numbering identifies the order in which the residues are incorporated into the cyclic pentapeptide backbone of CMN. The 2,3-l-DAP tethered to the PCP domain of CmnI is proposed to be introduced by the A domain of CmnF, analogous to the mechanism proposed for the VIO NRPS (34). R, residue.