Abstract

Amycolatopsis rifamycinica DSM 46095 is an actinobacterium that produces rifamycin SV, an antibiotic used againstMycobacterium tuberculosis. Here, we present the draft genome of DSM 46095, which harbors a novel rifamycin polyketide biosynthetic gene cluster (rif PKS) that differed by 10% in nucleotide sequence from the already reportedrif PKS cluster ofAmycolatopsis mediterranei S699.

GENOME ANNOUNCEMENT

Amycolatopsis mediterranei DSM 46095 was first isolated from a soil sample in an arid region near Alice Springs, Australia (1). At the time of isolation, this strain was named asNocardia mediterranei (1), thenAmycolatopsis mediterranei (2), and finallyAmycolatopsis rifamycinica (3).A. rifamycinica DSM 46095 was reported to produce rifamycin SV (1), which is a semisynthetic derivative of rifamycin B and is used for curing tuberculosis.

The rifamycin polyketide synthase (rif PKS) gene cluster, involved in the synthesis of rifamycin B, was characterized from two strains ofA. mediterranei, S699 (4) and LBGA 3136 (5). While therif PKS gene clusters were essentially identical in both of these strains (4–6), the partially characterized (~4 kb)rif PKS-like gene cluster fromA. rifamycinica DSM 46095 showed 10% differences in nucleotide sequences compared to therif PKS clusters from S699 and LBGA 3136 (7). To further understand the novel rifamycin polyketide synthase gene cluster, the genome sequencing of this strain was performed.

Total genomic DNA of strain 46095 was sequenced by the Illumina Genome Analyzer platform IIx using a PCR-free-based approach (paired-end library, 2 kb [n = 12,157,214] and 500 bp [n = 1,4035,608]). The draft genome sequence (9.20 Mb) of strain 46095 was assembled (150× coverage) into 88 contigs (>500 bp [±10 bp]) using the ABySS 1.3.5 assembler (8) set at a k-mer size of 63. The final validated assembly (N₅₀ contigs, 491,147 bp) was annotated using RAST version 4.0 (9) and the NCBI Prokaryotic Genomes Annotation Pipeline (PGAP) (http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html). There were 8,307 coding sequences (CDSs), with an average GC content of 71.8%, 414 subsystems, and 83 pseudogenes. These CDSs fall into 6,879 functional clusters of orthologous groups (COGs) with the presence of 2,491 hypothetical proteins. By using the antiSMASH server (10), we observed ~82 secondary metabolite gene clusters encoding type I specific polyketide synthases (PKS), type II PKS, nonribosomal peptide synthetases (NRPS), hybrid PKS, lantipeptides, lycopenes, and terpenes in the draft genome.

Therif PKS gene cluster in strain 46095 was represented by a continuous fragment of ~85 kb on contig number 39 that containedrifA,rifB,rifC,rifD, andrifE open reading frames (ORFs). Acyltransferase (AT) and ketosynthase (KS) domains present on these ORFs showed 90% homology with that ofA. mediterranei S699 (4). Apart from a uniquerif PKS cluster, eight other PKS, seven NRPS, and three hybrid NRPS/PKS clusters were also present in the genome (11).

A complex regulatory network of 590 transcriptional regulators, including genes for 98 histidine kinases, 82 response regulators, and 1 phosphotransferase protein, was present. Fifty-six tRNAs, 18 rRNAs, 1,242 tandem repeats, and 2 clustered regularly interspaced short palindromic repeat (CRISPR) elements were also identified. Average nucleotide identity (ANI) (12) analysis revealed that the draft genome ofA. rifamycinica 46095 is phylogenetically related toAmycolatopsis mediterranei S699 (92.55%) (6),Amycolatopsis mediterranei U32 (92.53%) (13), andAmycolatopsis mediterranei RB (92.52%) (CP003777).

The sequence information of a novel rifamycin biosynthetic gene cluster ofA. rifamycinica 46095 will help in the analysis of the structure of polyketide being produced by this strain. This information, supplemented with other genomic information, might also be used to design combinatorial strategies like domain replacement and domain inactivation (14) to produce rifamycin analogs (15).

ACKNOWLEDGMENTS

The work was supported by grants from the Department of Biotechnology (DBT), Government of India, and the University of Delhi/Department of Science and Technology Promotion of University Research and Scientific Excellence DU DST—PURSE grant. A.S., R.K., U.M., and P.S. gratefully acknowledge the University Grants Commission, New Delhi, for providing research fellowships.

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