ORCID Profile
0000-0002-2637-3070
Current Organisation
UNSW Sydney
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Publisher: Wiley
Date: 16-05-2021
Abstract: We report evidence further supporting homology between proteins in the F 1 F O ‐ATP synthetase and the bacterial flagellar motor (BFM). BFM proteins FliH, FliI, and FliJ have been hypothesized to be homologous to F O ‐b + F 1 ‐δ, F 1 ‐α/β, and F 1 ‐γ, with similar structure and interactions. We conduct a further test by constructing a gene order dataset, examining the order of fliH , fliI , and fliJ genes across the phylogenetic breadth of flagellar and nonflagellar type 3 secretion systems, and comparing this to published surveys of gene order in the F 1 F O ‐ATP synthetase, its N‐ATPase relatives, and the bacterial/archaeal V‐ and A‐type ATPases. Strikingly, the fliHIJ gene order was deeply conserved, with the few exceptions appearing derived, and exactly matching the widely conserved F‐ATPase gene order atpFHAG , coding for subunits b‐δ‐α‐γ. The V/A‐type ATPases have a similar conserved gene order. Our results confirm homology between these systems, and suggest a rare case of synteny conserved over billions of years, predating the Last Universal Common Ancestor (LUCA).
Publisher: American Association for the Advancement of Science (AAAS)
Date: 25-11-2022
Abstract: Determining which cellular processes facilitate adaptation requires a tractable experimental model where an environmental cue can generate variants that rescue function. The bacterial flagellar motor (BFM) is an excellent candidate—an ancient and highly conserved molecular complex for bacterial propulsion toward favorable environments. Motor rotation is often powered by H + or Na + ion transit through the torque-generating stator subunit of the motor complex, and ion selectivity has adapted over evolutionary time scales. Here, we used CRISPR engineering to replace the native Escherichia coli H + -powered stator with Na + -powered stator genes and report the spontaneous reversion of our edit in a low-sodium environment. We followed the evolution of the stators during their reversion to H + -powered motility and used both whole-genome and RNA sequencing to identify genes involved in the cell’s adaptation. Our transplant of an unfit protein and the cells’ rapid response to this edit demonstrate the adaptability of the stator subunit and highlight the hierarchical modularity of the flagellar motor.
Publisher: Cold Spring Harbor Laboratory
Date: 27-01-2021
DOI: 10.1101/2021.01.26.427765
Abstract: Determining which cellular processes facilitate adaptation requires a tractable experimental model where an environmental cue can generate variants which rescue function. The Bacterial Flagellar Motor (BFM) is an excellent candidate – an ancient and highly conserved molecular complex for propulsion which navigates bacteria towards favourable environments. In most species, rotation is powered by H + or Na + ion transit through the torque-generating stator subunit of the motor complex. The ion that drives the rotor has changed over evolutionary timescales but the molecular basis of this selectivity remains unknown. Here we used CRISPR engineering to replace the native Escherichia coli H + -powered stator with Na + -powered stator genes and report the rapid and spontaneous reversion of our edit in a low sodium environment. We followed the evolution of the stators during their reversion to H + -powered motility and used whole genome and transcriptome sequencing to identify both flagellar- and non-flagellar-associated genes involved in the cell’s adaptation. Our transplant of an unfit protein and the cells’ rapid response to this edit demonstrates the adaptability of the stator subunit and highlights the hierarchical modularity of the flagellar motor.
Publisher: Cold Spring Harbor Laboratory
Date: 02-01-2021
DOI: 10.1101/2021.01.01.425057
Abstract: Evidence of homology between proteins in the ATP synthetase and the bacterial flagellar motor (BFM) has been accumulating since the 1980s. Specifically, the BFM’s Type 3 Secretion System (T3SS) export apparatus FliH, FliI, and FliJ are considered homologous to F O -b + F 1 -δ, F 1 -α/β, and F 1 -γ, and have similar structure and interactions. We review the discoveries that advanced the homology hypothesis and then conduct a further test by examining gene order in the two systems and their relatives. Conservation of gene order, or synteny, is often observed between closely related prokaryote species, but usually degrades with phylogenetic distance. As a result, observed conservation of synteny over vast phylogenetic distances can be evidence of shared ancestral coexpression, interaction, and function. We constructed a gene order dataset by examining the order of fliH , fliI , and fliJ genes across the phylogenetic breadth of flagellar and nonflagellar T3SS. We compared this to published surveys of gene order in the F 1 F O -ATP synthetase, its N-ATPase relatives, and the bacterial/archaeal V- and A-type ATPases. Strikingly, the fliHIJ gene order was deeply conserved, with the few exceptions appearing derived, and exactly matching the widely conserved F-ATPase gene order atpFHAG , coding for subunits b - δ - α - γ . The V/A-type ATPases have a similar conserved gene order shared for homologous components. Our results further strengthen the argument for homology between these systems, and suggest a rare case of synteny conserved over billions of years, dating back to well before the Last Universal Common Ancestor (LUCA).
Publisher: Oxford University Press (OUP)
Date: 2023
Abstract: The bacterial flagellar motor (BFM) is a rotary nanomachine powered by the translocation of ions across the inner membrane through the stator complex. The stator complex consists of two membrane proteins: MotA and MotB (in H+-powered motors), or PomA and PomB (in Na+-powered motors). In this study, we used ancestral sequence reconstruction (ASR) to probe which residues of MotA correlate with function and may have been conserved to preserve motor function. We reconstructed 10 ancestral sequences of MotA and found four of them were motile in combination with contemporary Escherichia coli MotB and in combination with our previously published functional ancestral MotBs. Sequence comparison between wild-type (WT) E. coli MotA and MotA-ASRs revealed 30 critical residues across multiple domains of MotA that were conserved among all motile stator units. These conserved residues included pore-facing, cytoplasm-facing, and MotA–MotA intermolecular facing sites. Overall, this work demonstrates the role of ASR in assessing conserved variable residues in a subunit of a molecular complex.
Publisher: Frontiers Media SA
Date: 19-03-2021
Publisher: Frontiers Media SA
Date: 23-12-2020
DOI: 10.3389/FMICB.2020.625837
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 roton 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.
No related grants have been discovered for Yan Lin.