Spotlights represent environmental factors that contribute to survival. Coevolution of our healthy microbiome communities [9] and their beneficial effects on our own tissue function underscores the communication that exists between our polymicrobial communities and the host [10]. Understanding those interactions that help maintain community stability and contribute to healthy periodontal tissue structure and function as opposed to those like P. gingivalis that disrupt community structure and subsequent interactions with the host is an exciting area of study. References 1 Darveau, R.P. (2009) The oral microbial consortium’s interaction with the periodontal innate defense system. DNA Cell Biol. 28, 389–395 2 Darveau, R.P. (2010) Periodontitis: a polymicrobial disruption of host homeostasis. Nat. Rev. Microbiol. 8, 481–490
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3 Maekawa, T. et al. (2014) Porphyromonas gingivalis manipulates complement and TLR signaling to uncouple bacterial clearance from inflammation and promote dysbiosis. Cell Host Microbe 15, 768–778 4 Hajishengallis, G. et al. (2011) Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement. Cell Host Microbe 10, 497–506 5 Jiao, Y. et al. (2013) Induction of bone loss by pathobiont-mediated Nod1 signaling in the oral cavity. Cell Host Microbe 13, 595–601 6 Kumar, P.S. (2012) Smoking and the subgingival ecosystem: a pathogen-enriched community. Future Microbiol. 7, 917–919 7 Cornforth, D.M. and Foster, K.R. (2013) Competition sensing: the social side of bacterial stress responses. Nat. Rev. Microbiol. 11, 285–293 8 West, S.A. et al. (2006) Social evolution theory for microorganisms. Nat. Rev. Microbiol. 4, 597–607 9 Ley, R.E. et al. (2006) Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124, 837–848 10 Curtis, M.A. et al. (2011) The relationship of the oral microbiotia to periodontal health and disease. Cell Host Microbe 10, 302–306
Giant steps toward understanding a mycoplasma gliding motor Mitchell F. Balish Department of Microbiology, Miami University, Oxford, OH 45056, USA
Mycoplasma mobile carries out gliding motility using a novel motor whose proposed mechanism more closely resembles eukaryotic cytoskeletal motors than other bacterial ones. High-resolution microscopy and techniques that take advantage of the special properties of the mycoplasma cell reveal that this motor propels cells in steps of discrete size. Gliding motility, the smooth movement of individual cells across a surface, is a phenomenon observed in numerous types of bacteria, but the molecular mechanisms that drive this process are confusingly disparate. In their recent Proceedings of the National Academy of Sciences USA publication, Kinosita et al. [1] find that the gliding mechanism used by Mycoplasma mobile bears surprising resemblances to those of eukaryotic cytoskeletal motors. Cell motility is a biological challenge that has been solved multiple ways in distantly related lineages. For movement of or within eukaryotic cells, cytoskeletal motors are employed [2]. These motors, which include myosins, kinesins, and dyneins, interact with cytoskeletal filaments – microfilaments and microtubules – in such a way that the motors are propelled directionally along the filaments. While one end of the motor is associated with the filament, which constitutes a track, the other end is associated with cargo that may include proteins, RNA, chromosomes, or organelles, including the plasma membrane itself. Cycles of ATP binding, hydrolysis, and release by the motor proteins are coupled to changes in motor Corresponding author: Balish, M.F. ([email protected]). 0966-842X/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.06.005
conformation that cause the motor to move down the track. This movement occurs in discrete steps with sizes that correspond to the repeating structure of the filaments and to the biophysical properties of the motor protein itself. These individual steps can be observed and measured under experimental conditions in which interactions between the motor and its substrate are limited. Although prokaryotes contain cytoskeletal filaments that are evolutionarily related to those of eukaryotic cells, molecular motors with the same mechanistic relationship to these filaments are not observed in these cells. Instead, many bacteria use flagella – unrelated to the eukaryotic structures with the same name – and/or type IV pili to move through liquid or across surfaces [3]. Other bacterial cells exhibit the much smoother gliding motility. Interestingly, the distinct lack of homology among the genes and proteins that are associated with motility across phylogenetically distant groups of gliding bacteria indicates that they have evolved distinct mechanisms for this movement [3]. Among the gliding bacteria are the cell wall-lacking mycoplasmas, which are well known for being genomically reduced to the point where they are the smallest life forms capable of axenic growth in the laboratory [4]. M. mobile is a gliding mycoplasma species associated with infection of fish gills [5]. Its gliding motor includes a transmembrane adhesin, Gli349, which is present in about 450 copies that project as 50-nm spikes from the cell surface near the forward end of the cell [6]. Gli349 allows adherence to surfaces with molecules containing sialic acid, which is not only abundant on cell surfaces but also present on the serum proteins in the media in which these cells are grown in the lab [6]. The deposition of these proteins onto glass slides allows 429
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(i)
(ii)
~70 nm
(iv)
(iii)
(v)
TRENDS in Microbiology
Figure 1. Propulsion of Mycoplasma mobile by movement of a Gli349 adhesin molecule. (i) The Gli349 adhesin (red) extends from the M. mobile cell surface (black) and engages with a sialic acid molecule (blue square) on the substrate. (ii) As part of the ATP hydrolysis cycle of the intracellular motor (purple), the adhesin is caused to change conformation, swinging 70 nm to the left from its original position (gray). This moves the cell 70 nm to the right. (iii) The adhesin disengages from the substrate. (iv) The adhesin swings back to the right. (v) The adhesin binds to a sialic acid molecule, completing the cycle.
observation of motility in vitro. Motility of M. mobile is powered by ATP hydrolysis; not only does depletion of ATP using arsenate rapidly bring the cells to a halt [7], but, remarkably, detergent-permeabilized cells whose cytoplasmic contents have escaped can be made to move again by adding ATP to the solution in which they are incubated [8]. In a proposed mechanism for propulsion of M. mobile cells [6], ATP hydrolysis by an intracellular protein associated with Gli349 results in conformational changes along the length of Gli349, impacting its interaction with surfaceassociated sialic acid-containing molecules. The orientation of the motor allows forward movement, disengagement, and reengagement with another sialic acidcontaining molecule. The binding of multiple motors to the substrate limits the chance that the cell will diffuse away during disengagement of any individual motor. This mechanism sounds astonishingly similar to eukaryotic cytoskeletal motors, but with two important differences. First, the track is on the outside of the cell instead of the inside. Second, the track is not linear and therefore does not determine the trajectory of the motor. Nonetheless, if the M. mobile motor is conceptually like eukaryotic cytoskeletal motors, then discrete steps ought to be observable under the right conditions. To test this hypothesis by searching for these individual steps, Kinosita et al. needed to alter two critical aspects of the conditions under which motility was observed [1]. The first was to restrict the ATP available to the motor, slowing the cell down so that discrete steps could be observed and measured. The second was to limit the ability of the motor to interact with the substrate, so that ideally, cell movements carried out by single motors, rather than the collective action of hundreds, could be observed. The researchers accomplished this using their permeabilized 430
cell model, which enabled them to limit ATP, and by competitively inhibiting binding of cell surface-associated Gli349 to the substrate with the inclusion of a sialylated carbohydrate. Labeling cells with a fluorescent molecule, the researchers observed the trajectories of individual cells using high precision colocalization microscopy, allowing them to track very small movements. Under these ATP- and surface binding-limited conditions, they could see the stopping and starting that are characteristic of individual step events. These displacements measured 72 14 nm. They repeated this experiment with intact cells, and although they could not control the amount of ATP, they were able to observe a smaller number of individual steps of 69 11 nm. This step size is entirely consistent with swinging of the 50 nm long Gli349 spikes through some arc (Figure 1). Thus, like eukaryotic cytoskeletal motor proteins, the M. mobile motor moves in discrete steps, even if the track is unorganized, lacking the regularly repeating structure of the linear cytoskeletal filaments of eukaryotic cells. The specific nature of the physical changes to Gli349 and other components of the motor remains unknown, although evidence points to Gli349 having hinges along its length. A deeper understanding of the workings of this motor, together with the vast body of knowledge derived from the study of the eukaryotic cytoskeletal motors that have converged to operate under similar principles, will enable future researchers to design motors with specified properties for nanotechnological uses, as has already been demonstrated for living M. mobile cells [9]. Acknowledgments I would like to thank Steven Distelhorst for editing suggestions.
Spotlights References 1 Kinosita, Y. et al. (2014) Unitary step of gliding machinery in Mycoplasma mobile. Proc. Natl. Acad. Sci. U.S.A. 111, 8601–8606 2 von Delius, M. and Leigh, D.A. (2011) Walking molecules. Chem. Soc. Rev. 40, 3656–3676 3 Jarrell, K.F. and McBride, M.J. (2008) The surprisingly diverse ways that prokaryotes move. Nat. Rev. Microbiol. 6, 466–476 4 Fadiel, A. et al. (2007) Mycoplasma genomics: tailoring the genome for minimal life requirements through reductive evolution. Front. Biosci. 12, 2020–2029
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5 Kirchhoff, H. and Rosengarten, R. (1984) Isolation of a motile mycoplasma from a fish. J. Gen. Microbiol. 130, 2439–2445 6 Miyata, M. (2010) Unique centipede mechanism of mycoplasma gliding. Annu. Rev. Microbiol. 64, 519–537 7 Jaffe, J.D. et al. (2004) Energetics of gliding motility in Mycoplasma mobile. J. Bacteriol. 186, 4254–4261 8 Uenoyama, A. and Miyata, M. (2005) Gliding ghosts of Mycoplasma mobile. Proc. Natl. Acad. Sci. U.S.A. 102, 12754–12758 9 Hiratsuka, Y. et al. (2006) A microrotary motor powered by bacteria. Proc. Natl. Acad. Sci. U.S.A. 103, 13618–13623
431
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3 Maekawa, T. et al. (2014) Porphyromonas gingivalis manipulates complement and TLR signaling to uncouple bacterial clearance from inflammation and promote dysbiosis. Cell Host Microbe 15, 768–778 4 Hajishengallis, G. et al. (2011) Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement. Cell Host Microbe 10, 497–506 5 Jiao, Y. et al. (2013) Induction of bone loss by pathobiont-mediated Nod1 signaling in the oral cavity. Cell Host Microbe 13, 595–601 6 Kumar, P.S. (2012) Smoking and the subgingival ecosystem: a pathogen-enriched community. Future Microbiol. 7, 917–919 7 Cornforth, D.M. and Foster, K.R. (2013) Competition sensing: the social side of bacterial stress responses. Nat. Rev. Microbiol. 11, 285–293 8 West, S.A. et al. (2006) Social evolution theory for microorganisms. Nat. Rev. Microbiol. 4, 597–607 9 Ley, R.E. et al. (2006) Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124, 837–848 10 Curtis, M.A. et al. (2011) The relationship of the oral microbiotia to periodontal health and disease. Cell Host Microbe 10, 302–306
Giant steps toward understanding a mycoplasma gliding motor Mitchell F. Balish Department of Microbiology, Miami University, Oxford, OH 45056, USA
Mycoplasma mobile carries out gliding motility using a novel motor whose proposed mechanism more closely resembles eukaryotic cytoskeletal motors than other bacterial ones. High-resolution microscopy and techniques that take advantage of the special properties of the mycoplasma cell reveal that this motor propels cells in steps of discrete size. Gliding motility, the smooth movement of individual cells across a surface, is a phenomenon observed in numerous types of bacteria, but the molecular mechanisms that drive this process are confusingly disparate. In their recent Proceedings of the National Academy of Sciences USA publication, Kinosita et al. [1] find that the gliding mechanism used by Mycoplasma mobile bears surprising resemblances to those of eukaryotic cytoskeletal motors. Cell motility is a biological challenge that has been solved multiple ways in distantly related lineages. For movement of or within eukaryotic cells, cytoskeletal motors are employed [2]. These motors, which include myosins, kinesins, and dyneins, interact with cytoskeletal filaments – microfilaments and microtubules – in such a way that the motors are propelled directionally along the filaments. While one end of the motor is associated with the filament, which constitutes a track, the other end is associated with cargo that may include proteins, RNA, chromosomes, or organelles, including the plasma membrane itself. Cycles of ATP binding, hydrolysis, and release by the motor proteins are coupled to changes in motor Corresponding author: Balish, M.F. ([email protected]). 0966-842X/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.06.005
conformation that cause the motor to move down the track. This movement occurs in discrete steps with sizes that correspond to the repeating structure of the filaments and to the biophysical properties of the motor protein itself. These individual steps can be observed and measured under experimental conditions in which interactions between the motor and its substrate are limited. Although prokaryotes contain cytoskeletal filaments that are evolutionarily related to those of eukaryotic cells, molecular motors with the same mechanistic relationship to these filaments are not observed in these cells. Instead, many bacteria use flagella – unrelated to the eukaryotic structures with the same name – and/or type IV pili to move through liquid or across surfaces [3]. Other bacterial cells exhibit the much smoother gliding motility. Interestingly, the distinct lack of homology among the genes and proteins that are associated with motility across phylogenetically distant groups of gliding bacteria indicates that they have evolved distinct mechanisms for this movement [3]. Among the gliding bacteria are the cell wall-lacking mycoplasmas, which are well known for being genomically reduced to the point where they are the smallest life forms capable of axenic growth in the laboratory [4]. M. mobile is a gliding mycoplasma species associated with infection of fish gills [5]. Its gliding motor includes a transmembrane adhesin, Gli349, which is present in about 450 copies that project as 50-nm spikes from the cell surface near the forward end of the cell [6]. Gli349 allows adherence to surfaces with molecules containing sialic acid, which is not only abundant on cell surfaces but also present on the serum proteins in the media in which these cells are grown in the lab [6]. The deposition of these proteins onto glass slides allows 429
Spotlights
Trends in Microbiology August 2014, Vol. 22, No. 8
(i)
(ii)
~70 nm
(iv)
(iii)
(v)
TRENDS in Microbiology
Figure 1. Propulsion of Mycoplasma mobile by movement of a Gli349 adhesin molecule. (i) The Gli349 adhesin (red) extends from the M. mobile cell surface (black) and engages with a sialic acid molecule (blue square) on the substrate. (ii) As part of the ATP hydrolysis cycle of the intracellular motor (purple), the adhesin is caused to change conformation, swinging 70 nm to the left from its original position (gray). This moves the cell 70 nm to the right. (iii) The adhesin disengages from the substrate. (iv) The adhesin swings back to the right. (v) The adhesin binds to a sialic acid molecule, completing the cycle.
observation of motility in vitro. Motility of M. mobile is powered by ATP hydrolysis; not only does depletion of ATP using arsenate rapidly bring the cells to a halt [7], but, remarkably, detergent-permeabilized cells whose cytoplasmic contents have escaped can be made to move again by adding ATP to the solution in which they are incubated [8]. In a proposed mechanism for propulsion of M. mobile cells [6], ATP hydrolysis by an intracellular protein associated with Gli349 results in conformational changes along the length of Gli349, impacting its interaction with surfaceassociated sialic acid-containing molecules. The orientation of the motor allows forward movement, disengagement, and reengagement with another sialic acidcontaining molecule. The binding of multiple motors to the substrate limits the chance that the cell will diffuse away during disengagement of any individual motor. This mechanism sounds astonishingly similar to eukaryotic cytoskeletal motors, but with two important differences. First, the track is on the outside of the cell instead of the inside. Second, the track is not linear and therefore does not determine the trajectory of the motor. Nonetheless, if the M. mobile motor is conceptually like eukaryotic cytoskeletal motors, then discrete steps ought to be observable under the right conditions. To test this hypothesis by searching for these individual steps, Kinosita et al. needed to alter two critical aspects of the conditions under which motility was observed [1]. The first was to restrict the ATP available to the motor, slowing the cell down so that discrete steps could be observed and measured. The second was to limit the ability of the motor to interact with the substrate, so that ideally, cell movements carried out by single motors, rather than the collective action of hundreds, could be observed. The researchers accomplished this using their permeabilized 430
cell model, which enabled them to limit ATP, and by competitively inhibiting binding of cell surface-associated Gli349 to the substrate with the inclusion of a sialylated carbohydrate. Labeling cells with a fluorescent molecule, the researchers observed the trajectories of individual cells using high precision colocalization microscopy, allowing them to track very small movements. Under these ATP- and surface binding-limited conditions, they could see the stopping and starting that are characteristic of individual step events. These displacements measured 72 14 nm. They repeated this experiment with intact cells, and although they could not control the amount of ATP, they were able to observe a smaller number of individual steps of 69 11 nm. This step size is entirely consistent with swinging of the 50 nm long Gli349 spikes through some arc (Figure 1). Thus, like eukaryotic cytoskeletal motor proteins, the M. mobile motor moves in discrete steps, even if the track is unorganized, lacking the regularly repeating structure of the linear cytoskeletal filaments of eukaryotic cells. The specific nature of the physical changes to Gli349 and other components of the motor remains unknown, although evidence points to Gli349 having hinges along its length. A deeper understanding of the workings of this motor, together with the vast body of knowledge derived from the study of the eukaryotic cytoskeletal motors that have converged to operate under similar principles, will enable future researchers to design motors with specified properties for nanotechnological uses, as has already been demonstrated for living M. mobile cells [9]. Acknowledgments I would like to thank Steven Distelhorst for editing suggestions.
Spotlights References 1 Kinosita, Y. et al. (2014) Unitary step of gliding machinery in Mycoplasma mobile. Proc. Natl. Acad. Sci. U.S.A. 111, 8601–8606 2 von Delius, M. and Leigh, D.A. (2011) Walking molecules. Chem. Soc. Rev. 40, 3656–3676 3 Jarrell, K.F. and McBride, M.J. (2008) The surprisingly diverse ways that prokaryotes move. Nat. Rev. Microbiol. 6, 466–476 4 Fadiel, A. et al. (2007) Mycoplasma genomics: tailoring the genome for minimal life requirements through reductive evolution. Front. Biosci. 12, 2020–2029
Trends in Microbiology August 2014, Vol. 22, No. 8
5 Kirchhoff, H. and Rosengarten, R. (1984) Isolation of a motile mycoplasma from a fish. J. Gen. Microbiol. 130, 2439–2445 6 Miyata, M. (2010) Unique centipede mechanism of mycoplasma gliding. Annu. Rev. Microbiol. 64, 519–537 7 Jaffe, J.D. et al. (2004) Energetics of gliding motility in Mycoplasma mobile. J. Bacteriol. 186, 4254–4261 8 Uenoyama, A. and Miyata, M. (2005) Gliding ghosts of Mycoplasma mobile. Proc. Natl. Acad. Sci. U.S.A. 102, 12754–12758 9 Hiratsuka, Y. et al. (2006) A microrotary motor powered by bacteria. Proc. Natl. Acad. Sci. U.S.A. 103, 13618–13623
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