The Streptomyces leeuwenhoekii genome: de novo sequencing and assembly in single contigs of the chromosome, circular plasmid pSLE1 and linear plasmid pSLE2

Abstract

BackgroundNext Generation DNA Sequencing (NGS) and genome mining of actinomycetes and other microorganisms is currently one of the most promising strategies for the discovery of novel bioactive natural products, potentially revealing novel chemistry and enzymology involved in their biosynthesis. This approach also allows rapid insights into the biosynthetic potential of microorganisms isolated from unexploited habitats and ecosystems, which in many cases may prove difficult to culture and manipulate in the laboratory. Streptomyces leeuwenhoekii (formerly Streptomyces sp. strain C34) was isolated from the hyper-arid high-altitude Atacama Desert in Chile and shown to produce novel polyketide antibiotics.ResultsHere we present the de novo sequencing of the S. leeuwenhoekii linear chromosome (8 Mb) and two extrachromosomal replicons, the circular pSLE1 (86 kb) and the linear pSLE2 (132 kb), all in single contigs, obtained by combining Pacific Biosciences SMRT (PacBio) and Illumina MiSeq technologies. We identified the biosynthetic gene clusters for chaxamycin, chaxalactin, hygromycin A and desferrioxamine E, metabolites all previously shown to be produced by this strain (J Nat Prod, 2011, 74:1965) and an additional 31 putative gene clusters for specialised metabolites. As well as gene clusters for polyketides and non-ribosomal peptides, we also identified three gene clusters encoding novel lasso-peptides.ConclusionsThe S. leeuwenhoekii genome contains 35 gene clusters apparently encoding the biosynthesis of specialised metabolites, most of them completely novel and uncharacterised. This project has served to evaluate the current state of NGS for efficient and effective genome mining of high GC actinomycetes. The PacBio technology now permits the assembly of actinomycete replicons into single contigs with >99 % accuracy. The assembled Illumina sequence permitted not only the correction of omissions found in GC homopolymers in the PacBio assembly (exacerbated by the high GC content of actinomycete DNA) but it also allowed us to obtain the sequences of the termini of the chromosome and of a linear plasmid that were not assembled by PacBio. We propose an experimental pipeline that uses the Illumina assembled contigs, in addition to just the reads, to complement the current limitations of the PacBio sequencing technology and assembly software.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1652-8) contains supplementary material, which is available to authorized users.

Isolation and anti-HIV-1 integrase activity of lentzeosides A–F from extremotolerant lentzea sp. H45, a strain isolated from a high-altitude Atacama Desert soil

“…It is especially interesting that the new compounds, lentzosides A-F, are derived from a novel Lentzia strain as previously new specialized metabolites from Atacama Desert actinomycetes have come from novel Streptomyces strains, 7 notably from a deep seated 16S rRNA gene clade that can be equated with the species, Streptomyces leeuwenhoekii. [10][11][12][13][14][15][16] The genus Lentzia was validly published in 1995, 25 but to date does not appear to have been studied in terms of its natural product chemistry, though Lentzia sp. 7887 effects the biotransformation of FR901459, a novel derivative of cyclosporin.…”

“…C38 and the cell invasion inhibitor chaxapeptin from S. leeuwenhoekii strain C58. [10][11][12][13][14][15][16] As part of our ongoing program to investigate the extremobiosphere as a source of new natural products, we have focused our attention on Lentzea sp. strain H45 which was isolated from a high-altitude Atacama Desert soil and shown to produce a specific pattern of secondary metabolites based on its liquid chromatography MS (LCMS) profile and associated NMR data.…”

Molecular beacons to identify gifted microbes for genome mining

“…The most gifted strains could be targeted for complete genome sequencing, which is now feasible and relatively inexpensive by using a combination of Illumina and PacBio sequencing. 16,[52][53][54] By adding additional CP-TEs from other taxons, the CP-TE multi-probe approach can be adapted to survey draft genomes of other bacteria with large genomes, such as species of the Myxobacteria, Burkholderia and Photorhabdus, all of which have gifted members that encode multiple SMGCs. 16 …”

The ‘gifted’ actinomycete Streptomyces leeuwenhoekii

“…However, initial inspection of this sequence using Artemis (Rutherford et al 2000), and particularly the GC Frame Plot option contained within it (Bibb et al 1984), revealed many likely mis-assemblies in contigs containing modular type I polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes. The short-size of the quality reads obtained from high mol% GC DNA (streptomycetes generally possess a GC content of around 73 mol% G?C) using Illumina sequencing (generally less than 250 nt) hampers the correct assembly of the repetitive regions characteristic of these genes Gomez-Escribano et al 2015, 2016. We thus set out to establish a high-quality genome sequence of S. leeuwenhoekii C34 T by combining two next generation sequencing technologies: the second-generation Illumina MiSeq renowned for very accurate short nucleotide-reads and the third-generation Single Molecule, Real-Time (SMRT) sequencing platform from Pacific Biosciences (PacBio) that gives long, but less accurate, nucleotide-reads (in our experience with Streptomyces DNA, around 5 kb on Antonie van Leeuwenhoek average after quality filtering) .…”

“…Gene clusters for desferrioxamine E (sle44550 to sle44600) and ectoine (sle52690 to sle52700) biosynthesis were also identified using antiSMASH version 2.0 (Blin et al 2013;Gomez-Escribano et al 2015;Razmilic 2017).…”