Understanding protein interaction and turnover in the bacterial flagellar motor

Lead Research Organisation: University of Oxford
Department Name: Biochemistry

Abstract

The majority of bacterial species can move, many swimming. They use swimming to reach their best environment for growth and survival. For pathogenic bacteria that can be a wound, or the lung or gut wall, for beneficial bacteria it could be a root, while for many bacteria it might be a surface where they can grow as a slime protected colony. Swimming uses a mechanism unique to bacteria, making it a possible site for controlling bacterial activity such as colonisation. However, to control swimming behaviour, and thus colonisation, we need to know how swimming works. Swimming relies on the rotation of rigid, helical flagellar filaments, the diameter of just over a hundredth of a millimetre. It was initially thought the bacterial flagellar filament was rotated by a structure in the bacterial membrane that operated like a tiny, stable electric motor with a ring of proteins, rotor proteins, anchored to the helical filament, which rotate against a fixed ring of proteins (stators) anchored to the cell wall. As charged particles move through the stator proteins a change in local charge interactions between the stator and rotor proteins drives the rotor ring round, at speeds of up to 1300 rev per sec. While this general mechanism is correct, a few years ago we showed that the proteins within a rotating motor are not in fixed rings, but some of the proteins within the rings are swapping with free proteins even though the motor is still spinning-rather like changing rotating parts of a car engine while driving at full speed down the motorway. Indeed more recently we found that if you removed the driving force, the ion gradient, altogether the supposedly anchored ring of stator proteins simply diffuses away from the rotor into the bacterial membrane, returning to the rotor one at a time only when the driving force is reinstated. The motor can also "feel" outside forces on the motor and adds additional components as the external force increases, as happens when the environment gets more viscous, as in a mucous gut or lung wall or when encountering a surface, allowing rotation to continue. The bacterial motor is therefore continually remodelling to allow swimming under a range of conditions. It has been known for a while that unrelated proteins that change concentration or activity during different growth conditions can alter swimming behaviour. It now seems likely that while remodelling these other proteins can "slip" in to the motor and interact with motor proteins to either slow or stabilise the motor structure and alter activity. In this study we intend to (1) characterise the effect on motor structure of these non-motor proteins, (2) to identify the changes that occur in the motor structure when it rotates under different conditions and (3) compare these changes across very different species with motors showing a common core, but with different rotational behaviours. This will allow the development of a more complete understanding of the dynamics of motor proteins and protein:protein interaction that could lead to the design of molecules that will interfer with that rotation and prevent colonisation.

Technical Summary

The flagellar motor is probably the most complex bacterial structure. The transmembrane rotary motor uses the ion motive force to drive an extracellular helical filament at up to 1300 rps, pushing the cell towards an optimum environment for growth. As synthesis depends on sequential expression of ~50 genes, and the final structure spans from the cytoplasm to the exterior, intact motors have not been isolated and images of the complete motor rely on partial structures and electron microscopy. Over the past few years our work and that of others using in vivo analysis of the protein components in single motors has shown that the motors are not stable but constantly remodelling, even while rotating. The ring of stator proteins providing the peptidoglycan anchored ring against which the rotor spins are only held in place by the flow of ions, and even when the ion gradient is maximal, the 11 or so stators each exchange with a membrane pool every 60 sec. The number of engaged stators changes as the external load on the motor changes. The proteins on the cytoplasmic face of the rotor, connecting the motor to the chemosensory system are also exchanging, but only if the motor in rotating in one direction. Other studies have shown that unrelated proteins such as H-NS, fumarate reductase and c-diGMP bound YcgR can interact with and alter motor activity, probably gaining access during motor remodelling. In the grant we will characterise:(1) motor protein exchange in E.coli and R.sphaeroides under conditions where the external load and the driving force is altered; (2) the differences between different stator systems encoded in one species; (3) the behaviour of the outer membrane proteins in the L-P ring and identify whether local peptidoglycan remodels; (4) the effect on remodelling of the binding of non-motor proteins. Together these data will provide a more complete picture of motor dynamics and potentially allow the design of molecules to prevent rotation and colonisation.

Planned Impact

The immediate impact of this research will be to the general bacterial community, increasing our general understanding of the bacterial flagellar motor and the dynamics of large macromolecular protein complexes in general and the possible roles of that dynamic exchange in function.

Industry:However, in the long run the impact may well be on industrial and medical applications. Swimming bacteria use motility to reach sites for forming biofilms or for colonisation within a host. Our recent work showing increasing stator numbers as external forces increase, mimicking the increased viscosity of a mucous membrane or the interaction of a motor with a surface, has implications for surface sensing and, in the longer term may provide a route to help develop mechanisms to intervene and prevent surface colonisation. Biofilm formation is of major industrial interest, causing fouling on everything from pipes and oil rigs to medical implants and being positively used in the treatment of waste water. A great deal of resource is spent creating surfaces that cannot be colonised or removing biofilms from surfaces, but producing molecules that could render bacteria transiently non-motile would reduce the numbers reaching a surface. This is a long term possibility, but if initial studies prove interesting we will discuss appropriate partner industries through ISIS.

Eduction:The applicant is actively involved with local schools and is involved in a BBSRC-funded education programme with the Oxford Natural History Museum to bring aspects of research within the university to a wider public. I am developing an interactive display within the Museum to illustrate the bacterial flagellar motor, its general importance in the determining crop growth (nodule formation) and disease, but also illustrating the mechanism by which we study this nanomachine and the possibilities for exploitation.

Training: The postdoctoral RA will be trained in a very wide range of techniques, from single molecule imaging through to moecular biophysics, data analysis and molecular genetics. It will also include developing informatic and modelling skills and an understanding of bacterial physiology, beyone E.coli, and protein chemistry. This wide range of techniques will ensure the RA is suitable for a wide range of positions at the end of the project. All researcher employed within the department are encouraged to attend a range of training programmes and as part of an interdisciplinary grouping the RA will be exposed to a very wide range of different approaches to tackling biological questions.

Publications

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Di Paolo D (2016) Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences