Systematic insertional mutagenesis of a streptomycete genome: a link between osmoadaptation and antibiotic production

Abstract

The model organism Streptomyces coelicolor represents a genus that produces a vast range of bioactive secondary metabolites. We describe a versatile procedure for systematic and comprehensive mutagenesis of the S. coelicolor genome. The high-throughput process relies on in vitro transposon mutagenesis of an ordered cosmid library; mutagenized cosmids with fully characterized insertions are then transferred by intergeneric conjugation into Streptomyces, where gene replacement is selected. The procedure can yield insertions in upward of 90% of genes, and its application to the entire genome is underway. The methodology could be applied to many other organisms that can receive DNA via RK2/RP4-mediated intergeneric conjugation. The system permits introduction of mutations into different genetic backgrounds and qualitative measurement of the expression of disrupted genes as demonstrated in the analysis of a hybrid histidine kinase and response regulator gene pair, osaAB, involved in osmoadaptation in Streptomyces. The independently transcribed response regulator gene, osaB, is essential for osmoadaptation; when grown with supplementary osmolyte, an osaB mutant cannot erect aerial hyphae and produces up to fivefold greater antibiotic yields than the wild-type strain.

Figure 1

osaB is essential for osmoadaptation. Cultures were incubated for 7 d at 30°C. (i) S. lividans strains grown on R2YE containing 10.3% sucrose: (A) 1326, (B) AB101 containing pPM927, (C) AB101 containing pAB04, (D) AB101, (E) X0236; (ii) S. coelicolor strains grown on R2YE containing 10.3% sucrose: (A) M145, (B) AB201, (C) AB001 containing pPM927, (D) AB001 containing pAB04, (E) AB001; (iii) the same S. coelicolor strains grown on MS containing 25 mM KCl; and (iv) grown on MS with no supplementary osmolyte.

Figure 2

Structure of Tn5062 and the osaAB gene region. (A) Tn5062 is comprised of the following elements (not drawn to scale): two Tn5 mosaic ends (ME) flanking a three-frame translational stop and a consensus streptomycete ribosome-binding site (rbs) preceding a promoterless copy of eGFP, the apramycin resistance gene, aac3(IV), itself flanked by two T4 transcriptional terminators, and oriT(RK2). The locations of the two sequencing primers, EZL2 and EZR1, are indicated. (B) Arrows above the osaAB gene region indicate the location of representative Tn5062 insertions (1–7) and the Tn5493 insertion (x). Tn5062 insertions 1–6 in osaAB are all oriented to place eGFP under transcriptional control of osaAB promoters. S. coelicolor strain AB201 contains an insertion at position 1 (position 6281495 in the chromsome), and strain AB001 contains an insertion at position 5 (position 6285442). The extent of the fragment and the restriction sites used for cloning osaB and its promoter for complementation are indicated beneath the gene. This gene was initially cloned linked to aac3(IV) of the Tn5062 insertion in SCO5751 (insertion 7).

Figure 3

Antibiotic overproduction by the S. coelicolor osaB mutant AB001. Graphs of yields (the average of three independent experiments; standard deviations were <5% of the plotted values) at indicated time-points of (A) actinorhodin and γ-actinorhodin, and (B) undecylprodigiosin relative to cell density (OD₄₅₀) produced by submerged cultures grown in liquid R5 with or without 10.3% sucrose (S); (•) AB001 (+S); (▪) M145 (+S); (♦) AB001 (-S); (▴) M145 (-S).

Figure 4

Transcription of osaB. (A) Fluorescence and phase-contrast images of substrate mycelia after 24 h growth of S. coelicolor AB001 on R2YE medium (i) with or (ii) without 10.3% sucrose (Bar, 10 μm). (B) High resolution SI nuclease mapping of the osaB transcription start site. (i) RNA was isolated at the time points indicated from cultures of S. coelicolor M145 grown on R2YE medium containing 10.3% sucrose. (ii) The corresponding nucleotide sequence of the promoter region of osaB, indicating -35 and -10 sequences, transcriptional start (arrow), the probable ribosome-binding site (rbs), and translational start (TS).