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 reconstruction nodes to evaluate the potential of ancestral sequence reconstruction (ASR) methods for stator engineering and to test specific motifs previously hypothesized to be 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 Escherichia 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 had reported previously. Additionally, four among the thirteen reconstructed B-subunits were compatible with the A-subunit of Aquifex aeolicus and able to function in a sodium-independent manner. Overall, this work demonstrates the use of ancestral reconstruction to generate novel stators and quantify which residues are correlated with which ionic power source.
Highlights
Bacterial cells can move through liquids or over moist surfaces using rotating flagella to propel themselves in response to chemical stimulus, temperature and pH (Armitage, 2007; Jarrell and McBride, 2008; Gurung et al, 2020)
Following our network-based selection of refined sequences (Supplementary Figure 1), seven Vibrio strains remained in our phylogeny, and ASR1459 was the nearest parental-node to these seven strains
The bacterial flagellar motor is a nanomachine powered by the translocation of specific ions across the cell membrane (Sowa and Berry, 2008)
Summary
Bacterial cells can move through liquids or over moist surfaces using rotating flagella to propel themselves in response to chemical stimulus, temperature and pH (Armitage, 2007; Jarrell and McBride, 2008; Gurung et al, 2020). One of the largest molecular machines in bacteria, with a molecular mass of ∼11 MDa (Sowa and Berry, 2008), the BFM is present in a great variety of bacterial taxa from various habitats (Pion et al, 2013) It shares structural and amino acid sequence homology across a diverse range of taxa which suggests early ancestry and makes it a case study for investigating the origins of microbial motility (Mitchell and Kogure, 2006; Thormann and Paulick, 2010; Son et al, 2015). The binding and release of proton or hydronium ions by the universally conserved MotB aspartate residue allow the MotA to bind the neighboring FliG, which trigger the conformational change in the stator complex and generate torque (Deme et al, 2020; Santiveri et al, 2020)
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