Design and validation of a PCR screen for γ-butyrolactone-like regulatory systems in Streptomyces

Abstract

γ-butyrolactone and related signalling systems are found in Streptomyces and other actinobacteria where they control the production of secondary or specialized metabolites such as antibiotics. Genetic manipulation of these regulatory systems therefore leads to changes in the secondary metabolite profile of a strain and has been used to activate previously silent secondary metabolite gene clusters. However, there is no easy way to assess the presence of γ-butyrolactone-like systems in Streptomyces strains without whole-genome sequencing. We have therefore developed and tested a PCR screen that is able to detect homologues of the commonly co-located butenolide synthase and γ-butyrolactone receptor genes. This PCR screen could be employed for the screening of strain libraries to detect signalling systems without the necessity for whole-genome sequencing.

Keywords: Streptomyces; autoregulators; butenolides; furans; secondary metabolism; specialised metabolism; γ-butyrolactones.

Fig. 1.

Target genes, target motifs, and PCR results. (a) scbR and scbA genes in divergent orientation, with primer binding sites indicated by red arrows. (b) YFHF motif and surrounding amino acids in ScbR homologues, (c) ETxRQ motif and surroundings in ScbA homologues. (d) Agarose gel electrophoresis (1.7%) of PCR products, see Table 3 for identification of lanes. L=NEB 100 bp ladder.

Fig. 2.

MEME motif logo showing the ARE sequence.