A Directed Mutanagenesis Approach to Identify High Persister Alleles of hipA in Escherichia Coli

Research Category: Physical and LIfe Sciences
Presenter: Nicholas Waters

Persisters are a subpopulation of phenotypic variants in a bacterial population tolerant to bactericidal antibiotics. Studies have implicated toxin-antitoxins in persister formation. hipBA is a TA module, mutations in which increase persisters in Escherichia coli. We sequenced hipA from 450 clinical and environmental isolates of E. coli. We saw marked polymorphism in hipA sequences of these strains as compared to the wildtype, some of which increased persisters by 1000-fold. This lead us to the question: under which conditions in the environment would hipA accumulate mutations leading to higher antibiotic tolerance?

In yeast, Hsp90 chaperone folds damaged proteins, allowing mutations that might otherwise cause loss of function to remain viable. We suggest that persister formation is similarly aided by the HtpG chaperone, which has been proven to be active in folding kinases (like HipA) and is the prokaryotic homologue to Hsp90 based on substrate size.   Studies have shown that genes with a higher level of expression acquire mutations more frequently, as do genes on the chromosome’s lagging strand when compared to the leading strand. The hipBA locus is present on the lagging strand, so we propose that overexpression of htpG in the presence of an inducer of hipA would result in an accumulation of mutations within the hipBA locus, potentially resulting in a high persister phenotype. By comparing mutation rates in hipBA between wild-type, ΔhtpG strain, and an htpG over-expression strain under hipA-inducing growth conditions, we hope to identify conditions responsible for producing high persister hipA mutations.