Investigation of thein vitroandin vivoactivity of the type IV pilus extension ATPase BfpD

Abstract

18Type IV pili (T4Ps) are multifunctional protein fibers found in many bacteria and archaea. All T4P systems 19 have an extension ATPase, which provides the energy required to push structural subunits out of the 20 membrane. We previously reported that the BfpD T4P ATPase from enteropathogenic E. coli (EPEC) has 21 the expected hexameric structure and ATPase activity, the latter enhanced by the presence of the N-22 terminal cytoplasmic domains of its partner proteins BfpC and BfpE. In this study, we further 23 investigated the kinetics of the BfpD ATPase. Despite high purity of the proteins, the reported enhanced 24 ATPase activity was found to be from (an) ATPase(s) contaminating the N-BfpC preparation. 25Furthermore, although two mutations in highly conserved bfpD sites led to loss of function in vivo, the 26 purified mutant proteins retained some ATPase activity, albeit less than the wild-type protein. 27Therefore, the observed ATPase activity of BfpD was also affected by (a) contaminating ATPase(s). 28Expression of the mutant bfpD alleles did not interfere with BfpD function in bacteria that also 29 expressed wild-type BfpD. However, a similar mutation of bfpF, which encodes the retraction ATPase, 30 blocked the function of wild-type BfpF when both were present. These results highlight similarities and 31 differences in function and activity of T4P extension and retraction ATPases in EPEC. 32 33 34