Deciphering tuberactinomycin biosynthesis: isolation, sequencing, and annotation of the viomycin biosynthetic gene cluster

Abstract

The tuberactinomycin antibiotics are essential components in the drug arsenal against Mycobacterium tuberculosis infections and are specifically used for the treatment of multidrug-resistant tuberculosis. These antibiotics are also being investigated for their targeting of the catalytic RNAs involved in viral replication and for the treatment of bacterial infections caused by methicillin-resistant Staphylococcus aureus strains and vancomycin-resistant enterococci. We report on the isolation, sequencing, and annotation of the biosynthetic gene cluster for one member of this antibiotic family, viomycin, from Streptomyces sp. strain ATCC 11861. This is the first gene cluster for a member of the tuberactinomycin family of antibiotics sequenced, and the information gained can be extrapolated to all members of this family. The gene cluster covers 36.3 kb of DNA and encodes 20 open reading frames that we propose are involved in the biosynthesis, regulation, export, and activation of viomycin, in addition to self-resistance to the antibiotic. These results enable us to predict the metabolic logic of tuberactinomycin production and begin steps toward the combinatorial biosynthesis of these antibiotics to complement existing chemical modification techniques to produce novel tuberactinomycin derivatives.

FIG. 1.

Chemical structures of the TUB family of antibiotics. The numbers within the cyclic pentapeptide core identify the residue numbers noted in the text. The figure is as presented elsewhere (55), with modifications.

FIG. 2.

Schematic representation of the viomycin biosynthetic gene cluster. The center line denotes the 37 kb encoding all ORFs involved in viomycin biosynthesis, with the bars above and below the center line representing DNA present on cosmids pVIO-P8C8RH and pVIO-P2C3RG, respectively. Arrows above and below the center line identify the direction of transcription of ORFs. Coding of ORF biosynthetic function is as follows: grey, NRPS; black, l-2,3-diaminopropionate; white, l-2,3-diaminopropionate→β-ureidodehydroalanine; vertical lines, l-capreomycidine; horizontal lines, l-capreomycidine hydroxylation; right-slanted lines, β-lysine; left-slanted lines, resistance and activation; checkerboard pattern, regulation; waves, export.

FIG. 3.

Schematic representations of the biosynthetic pathways for the four nonproteinogenic amino acids in Vio. The abbreviations are defined in the text. In panel D, R is —H or —tripeptide, and the last step in β-ureidodehydroalanine biosynthesis occurs after cyclic pentapeptide synthesis.

FIG. 4.

Schematic representations of the viomycin NRPS. The domains of each subunit are shown as circles. The bars below the NRPS subunits denote specific modules, which are annotated M1 through M5. The arrow between VioF and VioI represents the in trans aminoacylation of VioI by the A1 domain of VioF. The grey arrows indicate the direction of peptide synthesis. The abbreviations for the NRPS domains are defined in footnote a of Table 1 and the text.

FIG. 5.

Schematic representation of VioO- and VioM-catalyzed N-acylation of des-β-lysine-viomycin with β-lysine.

FIG. 6.

(A) Schematic representation of the insertion of pOJ260-vioA into the chromosomal copy of vioA by single homologous recombination. Grey boxes, regions of identity between the cloned vioA fragment and vioA on the chromosome; arrowheads, locations of primers used to confirm the vioA::pOJ260-vioA mutations; black box and associated Apr^R, the apramycin resistance gene on pOJ260-vioA. Resistance to this antibiotic was used for selection of the single-crossover insertional inactivation of vioA. (B) Representative HPLC traces comparing the viomycin produced by a wild-type strain and one of the vioA-negative (vioA⁻) strains (strain MGT1001) of Streptomyces sp. strain ATCC 11861 to authentic viomycin (10 μg). Viomycin was not detected in vioA-negative strain MGT1002 or MGT1003 (data not shown).