Computational protocol: Complete Genome Sequence of Thiostrepton-Producing Streptomyces laurentii ATCC 31255

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Protocol publication

[…] Streptomyces species are Gram-positive aerobic mycelial bacteria belonging to the family Streptomycetaceae (). These bacteria have the ability to produce a wide variety of secondary metabolites, including antibiotics (), and are also interesting subjects with which to study morphological differentiation (). Pock formation involving conjugative plasmids is a typical physiological characteristic used for morphological differentiation of Streptomyces species (), which contain both plasmids and a large linear chromosome.Streptomyces laurentii ATCC 31255 was classified as a thiostrepton producer not involving Streptomyces azureus, Streptomyces hawaiiensis, or Streptomyces sp. strain X-14b (). It is distinct from the Streptomyces fradiae group (), and Streptomyces termitum and Streptomyces roseofulvus are contained within the S. laurentii group (). The thiostrepton resistance gene (tsr) is a selective marker often used in the genetic engineering of actinomycetes and was isolated from the strain (). We previously reported that S. laurentii contains a linear conjugative plasmid, pSLL (), and circular integrative plasmid, pSLS (). pSLL suppressed the injurious effects caused by pSLS, which include marked decreases in spore formation and thiostrepton productivity. The whole-genome sequence of S. laurentii may shed light on the mechanisms of spore formation and thiostrepton production and may also be useful for comparative studies of morphological and metabolic differentiation among thiostrepton-producing Streptomyces species.A sample was prepared for sequencing by growing S. laurentii ATCC 31255 aerobically overnight at 28°C in tryptic soy broth (TSB) (Oxoid). The genomic DNA was then extracted and purified, as we described previously (). The prepared genome was sequenced using the PacBio RSII platform; 150,292 raw reads resulted in 100,282 quality-filtered trimmed reads, yielding 741 Mb, with a mean genome-wide coverage of 77.43×. The filtered reads were assembled using HGAP version 2.3.0 () and resulted in a 1-contig scaffold. Annotation was performed using Microbial Genome Annotation Pipeline (MiGAP; genome of S. laurentii ATCC 31255 includes a linear chromosome, with a G+C content of 72.3%. Annotation using the COG, RefSeq, and TrEMBL databases with tRNAscan-SE version 1.23 and additional manual inspection revealed 7,452 predicted coding regions, 69 tRNA genes, and 7 rRNA genes.Terminal inverted repeats are contained at the end of the chromosome of S. laurentii. In addition, a gene cluster for thiostrepton biosynthesis was annotated from genes SLA3859 (tsrS) to SLA3877 (tsrA) (). This cluster shows strong similarity to the thiostrepton biosynthesis cluster of S. azureus (). The tsr gene is located between the EF-Tu gene and the N-acetylmuramoyl-l-alanine amidase family protein-coding gene. One copy of the pSLS sequence is integrated between SLA2930 and SLA2952, and a second copy is between SLA3776 and SLA3796. Plasmid pSLL does not exist in free form within its host cell. […]

Pipeline specifications

Software tools HGAP, tRNAscan-SE
Applications Genome annotation, WGS analysis
Organisms Streptomyces laurentii
Chemicals Thiostrepton