Gain- and Loss-of-Function Screens Coupled to Next-Generation Sequencing for Antibiotic Mode of Action and Resistance Studies in Streptococcus pneumoniae.

ABSTRACT: Two whole-genome screening approaches are described for studying the mode of action and the mechanisms of resistance to trimethoprim (TMP) in the Gram-positive Streptococcus pneumoniae The gain-of-function approach (Int-Seq) relies on a genomic library of DNA fragments integrated into a fucose-inducible cassette. The second approach, leading to both gain- and loss-of-function mutation, is based on chemical mutagenesis coupled to next-generation sequencing (Mut-Seq). Both approaches pointed at the drug target dihydrofolate reductase (DHFR) as a major resistance mechanism to TMP. Resistance was achieved by dhfr overexpression either through the addition of fucose (Int-Seq) or by mutations upstream of the gene (Mut-Seq). Three types of mutations increased expression by disruptin

SUBMITTER: Gingras H 

PROVIDER: S-EPMC6496052 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature