AUTHOR=Islam Md Imtiazul , Lin Angela , Lai Yu-Wen , Matzke Nicholas J. , Baker Matthew A. B. 

TITLE=Ancestral Sequence Reconstructions of MotB Are Proton-Motile and Require MotA for Motility

JOURNAL=Frontiers in Microbiology

VOLUME=Volume 11 - 2020

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.625837

DOI=10.3389/fmicb.2020.625837

ISSN=1664-302X

ABSTRACT=The Bacterial flagellar motor (BFM) is a nanomachine that rotates the flagellum to propel many known bacteria. The BFM is powered by ion transit across the cell membrane through the stator complex, a membrane protein. Different bacteria use various ions to run their BFM, but the majority of BFMs are powered by either proton (H+) or sodium (Na+) ions. The transmembrane (TM) domain of the B-subunit of the stator complex is crucial for ion selectivity, as it forms the ion channel in complex with TM3 and TM4 of the A-subunit. In this study, we reconstructed and engineered thirteen ancestral sequences of the stator B-subunit to evaluate the functional properties and ionic power source of the stator proteins at nodes where known contemporary sodium and proton swimmers diverged to inform motifs involved in ion-selectivity. We found that all thirteen of our reconstructed ancient B-subunit proteins could assemble into functional stator complexes in combination with the contemporary E. coli MotA-subunit to restore motility in stator deleted E. coli strains. The flagellar rotation of the thirteen ancestral MotBs was found to be Na+ independent which suggested that the F30/Y30 residue was not significantly correlated with sodium/proton phenotype, in contrast to what we reported previously. Additionally, four among the thirteen reconstructed B-subunits were compatible with the A-subunit of Aquifex aeolicus and also functioned independently of sodium. Overall, this work uses ancestral sequence reconstruction to generate novel stators and quantify which residues correlate with which ionic power source.