Deletion and Characterization of Two Signaling Genes in Streptomyces

Abstract

Streptomyces coelicolor is a filamentous, high G‐C (guanine‐cytosine), gram‐positive bacterium that produces an aerial mycelium. The genome consists of a 8,667,507 base pair linear chromosome for this organism and contains the largest number of genes so far discovered in a bacterium. It belongs to the phylum Actinobacteria. In addition, streptomycetes are used to produce the majority of antibiotics in the medical field. This study focuses on the effect of the deletion of Sco5218, which encodes a putative integral membrane protein and Sco5219, which encodes a putative lipoprotein in Streptomyces coelicolor. Both of these genes are found in a potential operon. The goal of this research is to construct the double mutant and discern differences in phenotype compared to the single deletion mutants of each gene. We hypothesize that the double mutant will have an enhanced effect on the sporulation defect of Streptomyces. Sco5218 is believed to contain an HD‐GYP domain through bioinformatics analysis and is highly conserved throughout the Streptomyces genus. The HD‐GYP domain is typically associated with cyclic di‐GMP specific phosphodiesterase activity. Following REDIRECT protocol, the Streptomyces coelicolor cosmid clone was introduced into E. coli via electroporation. PCR was then utilized for the deletion of Sco5218 and Sco5219. This PCR product was run onto a gel and purified. This pure PCR product was then transformed into the plasmid vector which will then be mated into Streptomyces coelicolor. The relevance of this research allows us to see the importance of the HD‐GYP domain in bacteria. We witnessed a slight delay in sporulation with the Sco5218 mutant deletion. With the added deletion of Sco5219 we hypothesize that there will be an enhanced delay in sporulation. If we witness a delay in the sporulation with the double mutant in Streptomyces we can deduce that both genes play a role in bacterial development. These findings will allow us to further delve into the study of other bacteria and have a basis for other orthologs containing the HD‐GYP domain.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.