Development of a conditional lethal system for a Streptomyces lividans strain and its use to investigate conjugative transfer in soil

Abstract

In Streptomyces lividans, a conditional lethal mechanism was obtained by inactivation of gylB in the glycerol operon. The resulting strain was efficiently counter-selected on glycerol-containing medium but the mutation had no effect on the capacity of the strain to be maintained in non-sterile soil samples and to transfer derivatives of the genetic element pSAM2. However, despite the efficiency of the donor counterselection system, the transfer of a pSAM2 derivative from S. lividans to indigenous soil bacteria was not detected. ß

Fine-tuning the regulation of Cas9 expression levels for efficient CRISPR-Cas9 mediated recombination in Streptomyces

“…This operon comprises four genes: gylA (glycerol uptake protein), gylB (glycerol kinase) and gylC (glycerol-3-phosphate dehydrogenase) are dedicated to the uptake and catabolism of glycerol, while gylR encodes a regulatory protein. Inactivation of gylB has been shown to be lethal for S. lividans when grown on a glycerolcontaining medium [35], allowing a screening of CRISPR-Cas9 mediated disruption of the glycerol uptake operon by growth phenotype with and without glycerol. Thus, plasmid pCMU-4dGly was built as described in "Methods" in order to delete a fragment of the chromosome containing 1202 nucleotides of gylB coding region and the first 402 nucleotides of the gylC coding sequence (Fig.…”