Draft Genome Sequence of Streptomyces clavuligerus NRRL 3585, a Producer of Diverse Secondary Metabolites

Abstract

Streptomyces clavuligerus is an important industrial strain that produces a number of antibiotics, including clavulanic acid and cephamycin C. A high-quality draft genome sequence of the S. clavuligerus NRRL 3585 strain was produced by employing a hybrid approach that involved Sanger sequencing, Roche/454 pyrosequencing, optical mapping, and partial finishing. Its genome, comprising four linear replicons, one chromosome, and four plasmids, carries numerous sets of genes involved in the biosynthesis of secondary metabolites, including a variety of antibiotics.

Streptomyces clavuligerus is a bacterium of industrial and clinical importance producing the β-lactamase inhibitor clavulanic acid (4), as well as cephamycin C (a β-lactam), clavams, tunicamycin, and holomycin (5, 9, 14, 17). S. clavuligerus is intriguing in regard to its genetic structure and mechanism of cephamycin C, clavulanic acid, and clavam biosynthesis (19, 20, 21). Here, we present a draft genome sequence of the type strain NRRL 3585 (ATCC 27064). The specific clone used, SC2, is a single-spore isolate from the stock of NRRL 3585 that showed good sporulation and metabolite production, a typical example of the wild-type strain.

The genome sequence was determined by Sanger paired-end sequencing (7) and Roche/454 pyrosequencing (12). Sanger reads at 4.9-fold coverage were produced from 4- or 40-kb genomic libraries, followed by 454 reads at 61.6-fold coverage. Sanger reads (14-fold coverage) provided by the Broad Institute were also utilized. The Sanger paired-end reads and the Newbler-assembled 454 contigs were assembled with a PCAP assembler (8). Optical mapping (Opgen, Inc.) was performed to confirm the assembly output and to assign contigs into each replicon. Gap closure was attempted using gap-spanning clones and PCR products. Coding sequences were predicted by the combined use of Glimmer (6), GeneMark (3), and CRITICA (2). Automatic functional annotation results obtained by AutoFACT (10) and the Rapid Annotation using Subsystem Technology (RAST) server (1) were compiled and validated with Artemis.

The genome consists of one linear chromosome (58 contigs in 4 scaffolds, 6,736,475 bp, 72.69% G+C) and four linear plasmids, pSCL1 (3 contigs in 2 scaffolds, 10,266 bp, 71.96% G+C), pSCL2 (2 contigs in 2 scaffolds, 149,326 bp, 70.07% G+C), pSCL3 (15 contigs in 2 scaffolds, 442,792 bp, 70.77% G+C), and pSCL4 (11 contigs in 3 scaffolds, 1,796,117 bp, 71.85% G+C). The 6.7-Mb chromosome is the smallest of the completely sequenced Streptomyces species. At least six rRNA operons and 66 tRNA genes as well as 7,898 protein-coding genes were annotated. Recently, a draft sequence of S. clavuligerus ATCC 27064 describing the 1.8-Mb megaplasmid was reported (13). The sequences of the chromosome and pSCL4 were nearly identical to our sequences. However, three other plasmids (15, 22) are present only in our data, suggesting that our clone has preserved the genome in its intact form.

A plethora of genes related to biosynthesis of secondary metabolites were discovered. The super-cluster for cephamycin C and clavulanic acid (21) is located on the chromosome, and the clavam cluster (19) is located approximately 1.4 Mb away. In contrast, a paralogue cluster (19) for clavulanic acid and clavam production lies on pSCL4. Dozens of gene sets for nonribosomal peptide synthetases, polyketide synthases, and the hybrids were detected from sequences of pSCL3, pSCL4, and the chromosome. Notably, two gene clusters that can synthesize enediyne-containing compounds, potent antitumor agents (11), were found. The genome also bears many gene clusters for secondary metabolites, such as staurosporine (18), moenomycin (16), terpenes, pentalenenes, phytoenes, siderophores, and lantibiotics.

This work could provide a platform to exploit bioactive compounds produced by S. clavuligerus NRRL 3585 and subsequently to develop strains better producing the molecules.

Nucleotide sequence accession numbers.

The assembled genome sequence of S. clavuligerus NRRL 3585 was deposited in GenBank under accession number ADWJ00000000, and the one described in this paper is the first version, ADWJ01000000. The sequence and annotation are also available from the Genome Encyclopedia of Microbes (GEM; http://www.gem.re.kr).