pWHM3

Streptomyces coelicolor is a filamentous soil bacterium that produces a variety of antibiotics. Streptococcus coelicolor abs8752 is an abs (antibiotic synthesis defective) type mutation in the absR locus. It is characterized by its inability to produce any of the four antibiotics synthesized by its parent strain, J1501. A chromosomal DNA fragment from Streptococcus coelicolor J1501 that was able to complement the Ab phenotype of the Abs8752 mutant was cloned and analyzed. DNA sequencing revealed the presence of two complete ORFs (SCO6992 and SCO6993) in the clone, in opposite directions. Introduction of SCO6992 into the mutant strain resulted in a significant increase in the production of two pigment antibiotics (actinomycin and undecylprodigiosin) in S. coelicolor J1501 and Abs8752. However, the introduction of SCO6993 did not show any significant difference compared to the control, suggesting that SCO6992 is mainly involved in stimulating antibiotic biosynthesis in S. coelicolor.

S.coelicolor J1501 was cultured in R2YE medium and used for chromosomal DNA isolation. To clone the absR complementary DNA fragment, the chromosomal DNA was partially digested with the restriction enzyme Sau3A1 and separated by electrophoresis on a 1% agarose gel. A pool of 4-6 kb DNA fragments recovered from the gel was ligated with the pWHM3 vector digested with BamHI and a mini-library was constructed by shotgun cloning. The ligated plasmid was transformed into Streptococcus coelicolor abs8752. Among 1,650 transformants, 1 colony with abs+ phenotype (pigment production) was selected to isolate the recombinant plasmid pWHM3-α3. pWHM3-α3 was able to restore actinomycin and undecylprodigiosin biosynthesis in S. coelicolor Ab8752 on R2YE plates (Figure 1A). This result strongly supports the ability of the cloned fragment to complement the pleiotropic antibiotic production defects of S. coelicolor abs8752.

Figure 1. Effect of cloned DNA fragments on pigmented antibiotic production in S. coelicolor. (Jin, Xue-Mei, et al., 2021)

When comparing the amounts of actinomycin and undecylprodigiosin produced in R2YE liquid medium on day 10,  it was seen that S. coelicolor abs8752/pWHM3-O1 produced 24.2 times more actinorhodin than transformants with either pWHM3 or pWHM3-O2, and 8.6 times more actinorhodin than that produced by S. coelicolor abs8752/pWHM3-α3 (Figure 1B, left). In addition, the amount of undecylprodigiosin produced by S. coelicolor abs8752/pWHM3-O1 was slightly higher than that produced by S. coelicolor abs8752/pWHM3-α3, and at least 4 times higher than those of the transformants with either pWHM3 or pWHM3-O2 (Figure 1B, right). This fact clearly demonstrates that the SCO6992 gene is responsible for inducing the biosynthesis of actinomycin and undecylprodigiosin in S. coelicolor strains.