Transposon insertion sequencing reveals novel hypermutator genes in .

Mutation rates in bacteria are an important determinant of adaptation to new environments and success in different niches. In some bacterial pathogens, "hypermutator" variants-most often associated with mutations in components of the DNA mismatch repair system-are associated with increased antibiotic resistance and poorer patient outcomes. We report the serendipitous finding of novel hypermutator genes in through genome-scale mutant fitness screening. Exposure of a transposon insertion mutant library of to extended weak antibiotic selection resulted in selection for mutations that directly increased fitness as expected, but also revealed genes where transposon insertion indirectly increased fitness due to elevated general mutation rates. Three novel hypermutator genes were confirmed in , encoding a transcription antiterminator; ABUW_0208, encoding a hypothetical protein; and ABUW_2121, which encodes a sulfite transporter. We find selection for hypermutator variants in transposon insertion sequencing (TIS) data sets from diverse bacteria under various antibiotic treatments. Our results expand the range of biological functions linked to hypermutator phenotypes in bacteria and provide a workflow for the identification of putative hypermutators by TIS.IMPORTANCEAll organisms have the capacity for evolution through mutation. Bacteria with high mutation rates have a survival advantage in some stressful environments because they generate beneficial mutations more frequently. "Hypermutators" are bacterial strains that carry gene inactivations that increase general mutation rates. These variants are important in chronic infections, as their increased genetic diversity allows higher drug resistance and prolonged survival in the host. Only a few different hypermutator genes are known, and there is no high-throughput method for their identification. We have made the serendipitous finding that hypermutator genes can be identified by genome-wide mutant fitness screening under specific selection conditions. We have identified novel hypermutator alleles in the notorious hospital pathogen and show that hypermutator variants can be detected in screens of a wide range of pathogens.