Identification and characterization of mutations in nmpR that restore type-IV pili dependent motility in Myxococcus xanthus

Abstract

Myxococcus xanthus is a Gram-negative bacterium that displays Type-IV pili dependent motility. These pili are transcriptionally regulated by the two-component system (TCS) PilSR. A strain of M. xanthus in which the response regulator (RR) pilR has been deleted is non-motile. However, after extended incubation, motility is restored due to mutations in a different TCS, NmpRSTU. Some of these identified mutations were in the RR nmpR and restored motility by causing NmpR to be in a constitutively active, or “ON” state. We sought to increase the number of known mutations in nmpR that would lead to this “ON” state and additionally characterize how these variants bind DNA compared to the wild type NmpR. A screen for nmpR mutants with restored motility was performed, and sequencing determined the site of mutation. Several mutations were identified in a linker domain of NmpR. To examine the impact of these mutations, NmpRON variants were constructed, purified, and analyzed using electromobility shift assays (EMSAs) to characterize DNA binding. It was observed that the NmpR variants had larger shifts compared to wildtype NmpR, even when one of two NmpR-binding sites on the DNA was mutated. This suggests that the NmpRON variants have increased oligomerization compared to the wildtype, which may explain how they are constitutively active. Since RRs are conserved across many bacterial species, this mutational analysis gives broad insight into how TCS can compensate for loss-of-function mutations.