Bacterial motility and chemotaxis,
and control of cellular protein positioning
Co-workers: Sheng-Wen Chiu, Andreas Diepold, Nelly Dubarry, Christopher Jones,
Matthew Smith, Andrea Szollossi, Stuart Thomas, Madhvi Venkatesh, Elaine Byles
We investigate bacterial motility and its control by environmental signals, and the mechanisms involved in positioning related large multiprotein complexes during the cell cycle, ensuring balanced responses.
We use a wide range of biochemical and biophysical techniques including single-molecule fluorescence microscopy and optical tweezers, bioinformatics and computer modelling and we work together closely with other groups within the Department of Biochemistry, the Department of Physics and external collaborators.
Our current research foci are:
- Principles of bacterial chemotaxis. Bacteria control the frequency of switching the rotational direction of their flagella to bias swimming in a favourable direction. Many bacteria use more than one pathway and must integrate signals from these pathways (Fig. 2). We combine molecular genetics, biochemistry, in vivo imaging and modelling to understand how signals integrate. R. sphaeroides also encodes multiple homologues of sensory proteins and we are using structural approaches, NMR, biophysical methods and crystallography to understand specificity.
- Chemoreceptor positioning and segregation in Rhodobacter sphaeroides. The two chemosensory pathways of R. sphaeroides are physically separate, one with membrane chemoreceptors in large arrays in the membrane, the other in a large cytoplasmic array, associated with the chromosome (Fig. 3). The cytoplasmic array uses a homologue, PpfA, of the large plasmid segregating ATPase, ParA to piggy-back on the segregating chromosome, ensuring each daughter inherits a cluster. We are using single molecule imaging, molecular genetics, biophysics and modelling to understand the mechanisms used to position and segregate the two pathways.
- Spatiotemporal control of the Rhodobacter cell cycle. R.sphaeroides has two chromosomes, each using a Par-like system. Using molecular genetics and fluorescent imaging we are developing a spatio-temporal understanding of the choreography of chromosome segregation relative to the positioning of the divisiome protein, FtsZ (Fig. 4).
- The bacterial flagellar motor and the Type III secretion system.The flagellar motor and the evolutionary related Type III secretion system (T3SS) (Fig. 5) are flexible and adaptive nanomachines. We use fluorescence microscopy including FRAP and single molecule measurements to investigate the activity of the motor and T3SS in vivo and have shown that both functioning nanomachines undergo adaptive remodelling in response to intra- and extra-cellular signals. Individual MotAMotB stator proteins exchange with pools of proteins every 30 sec, and the number of stators depends on both the proton gradient across the membrane and the external force of the flagellar filament (Fig. 6). The switch proteins also exchange and this exchange is linked to the direction of motor rotation. These studies are now being extended to investigate stator dynamics in other bacterial species including Pseudomonas aeruginosa and Shewanella onedensis and to investigate the impact of adaptive remodelling on other cellular processes
- Paulick A, Delalez NJ, Brenzinger S, Steel BC, Berry RM, Armitage JP & Thormann KM (2015) Dual stator dynamics in the Shewanella oneidensis MR-1 flagellar motor. Mol. Microbiol. doi: 10.1111/mmi.12984
- Diepold A, Kudryashev M, Delalez NJ, Berry RM, Armitage JP (2015) Composition, Formation, and Regulation of the Cytosolic C-ring, a Dynamic Component of the Type III Secretion Injectisome. PLoS Biol. 2015 Jan 15;13(1):e1002039. doi: 10.1371/journal.pbio.1002039.
- Kuchma SL, Delalez NJ, Filkins LM, Snavely EA, Armitage JP, O'Toole GA (2015) Cyclic Di-GMP-Mediated Repression of Swarming Motility by Pseudomonas aeruginosa PA14 Requires the MotAB Stator. J Bacteriol. 2015 Feb 1;197(3):420-30. doi: 10.1128/JB.02130-14.
- Kudryashev M, Diepold A, Amstutz M, Armitage JP, Stahlberg H & Cornelis GR (2015) Yersinia enterocolitica type III secretion injectisomes form regularly spaced clusters, which incorporate new machines upon activation. Mol. Microbiol. 95: 875-884. doi: 10.1111/mmi.12908.
- Popp F, Armitage JP, Schüler D (2014) Polarity of bacterial magnetotaxis is controlled by aerotaxis through a common sensory pathway. Nat Commun. 2014 Nov 14;5:5398. doi: 10.1038/ncomms6398.
- Delalez NJ, Berry RM, Armitage JP (2014) Stoichiometry and turnover of the bacterial flagellar switch protein FliN. MBio. 2014 Jul 1;5(4):e01216-14. doi: 10.1128/mBio.01216-14.
- Rosser G, Baker RE, Armitage JP, Fletcher AG (2014) Modelling and analysis of bacterial tracks suggest an active reorientation mechanism in Rhodobacter sphaeroides. J R Soc Interface. 2014 Aug 6;11(97):20140320. doi: 10.1098/rsif.2014.0320
- Briegel, A, Ladinsky MS, Oikonomou C, Jones CW,. Harris MJ, Fowler DJ, Chang Y-W, Thompson LK, Armitage JP and Jensen GJ (2014) Structure of bacterial cytoplasmic chemoreceptor arrays and implications for chemotactic signaling eLife 3:e02151. DOI: 10.7554/eLife.02151
- Rosser, G, Baker, RE, Armitage, JP, Fletcher, AG (2014) Modelling and analysis of bacterial tracks suggest an active reorientation mechanism in Rhodobacter sphaeroides J Roy Soc Interface (in press)
Figure 1: Research impressions from the Armitage lab
Clockwise from left upper corner: R. sphaeroides with polar flagellum; membrane chemoreceptor array (blue) and cytoplasmic array (green) in R. sphaeroides; Membrane and cytoplasmic receptors and sensory proteins both form hexagonal arrays; traces of static (red) and dynamic (blue) C-ring proteins in the Yersinia enterocolitica Type III secretion system, using PALM. Centre: Z-ring (red) and membrane chemoreceptor array (green) in R. sphaeroides
Figure 2: The Rhodobacter sphaeroides chemotaxis pathways.
The chemosensory proteins that control the flagellum form two distinct signalling clusters. The transmembrane chemoreceptors sense periplasmic concentrations of attractants, while the cytoplasmic chemoreceptors detect the metabolic state of the cell. (adapted from Porter et al., 2011)
Figure 3: Kymographs of TlpT-YFP showing the cytoplasmic cluster splitting.
A-C: TlpT-YFP fluorescent signal in cells in which the formation of two clusters is seen. Horizontal scale bar is 1 µm, vertical scale bar is 10 min. D: Schematic representation of the corresponding stage of the division cycle
Figure 4: The development process of the Z-ring and localisation of the replication origin of the two chromosomes of R. sphaeroides..
A. Time-lapse images of the cell cycle stage-specific localization of FtsZ in R. sphaeroides. The completion of septation results in two FtsZ spots at the new poles. Red: FtsZ-YFP; blue: differential interference contrast. Numbers: minutes. B. Z-ring precursors. (i) Polar FtsZ-YFP spots move to the future cytokinetic sites (30'-60') and FtsZ gradients extend from the midcell nodes (60'-90'). (ii) Time-lapse images of the midcell showing the FtsZ gradient (dashed arrows) between two midcell nodes. (Chiu et al., 2013) C / D. Localisation of the origin of chromosome 1 / 2. Left panels show representative images of epifluorescence in live cells, the origin is visualized through the binding of its own ParB protein fused to YFP. Right panels show the corresponding dotplots
Figure 5: Schematic representations of the flagellar motor and the injectisome
Figure 6: The number of stator units increases with growing external force on the flagellum.
Fluorescence intensity, which reflects the number of stators, increases with external load in both clockwise (CW) and counterclockwise (CCW) locked cells