A Molecular Clutch Disables Flagella in the
            <i>Bacillus subtilis</i>
            Biofilm

Blair, Kris M.; Turner, Linda; Winkelman, Jared T.; Berg, Howard C.; Kearns, Daniel B.

doi:10.1126/science.1157877

Cited by 298 publications

(319 citation statements)

References 24 publications

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“…Using the cell body as a handle for an optical trap we estimated that the brake can resist a torque up to 2-3 times the stall torque of the motor. In contrast, recently Blair et al showed that Bacillus subtilis uses a molecular clutch to disable its flagella during biofilm formation (19). Tethered cells overexpressing the clutchinitiating protein, EspE, behaved as although they were unpowered, analogous to a strain lacking torque-generating units (⌬motAmotB).…”

Section: Discussionmentioning

confidence: 99%

A molecular brake, not a clutch, stops the Rhodobacter sphaeroides flagellar motor

Pilizota

Brown

Leake

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Many bacterial species swim by employing ion-driven molecular motors that power the rotation of helical filaments. Signals are transmitted to the motor from the external environment via the chemotaxis pathway. In bidirectional motors, the binding of phosphorylated CheY (CheY-P) to the motor is presumed to instigate conformational changes that result in a different rotor-stator interface, resulting in rotation in the alternative direction. Controlling when this switch occurs enables bacteria to accumulate in areas favorable for their survival. Unlike most species that swim with bidirectional motors, Rhodobacter sphaeroides employs a single stopstart flagellar motor. Here, we asked, how does the binding of CheY-P stop the motor in R. sphaeroides-using a clutch or a brake? By applying external force with viscous flow or optical tweezers, we show that the R. sphaeroides motor is stopped using a brake. The motor stops at 27-28 discrete angles, locked in place by a relatively high torque, approximately 2-3 times its stall torque.

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Section: Discussionmentioning

confidence: 99%

A molecular brake, not a clutch, stops the Rhodobacter sphaeroides flagellar motor

Pilizota

Brown

Leake

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…The amphiphilic nature of this molecule hinders it from freely crossing the membrane into the cellular interior except through the endocytic pathway, as extensively characterized in eukaryotic cells (30,31). FM 4-64 has also been widely used in bacterial cells and shown to specifically label membranes but not extracellular protein filaments such as flagella (32,33), except in a few bacterial species where flagella are coated in membrane sheaths (34). To our surprise, the entire length of the Shewanella nanowires was clearly stained with this reliable lipid bilayer dye (Fig.…”

Section: Resultsmentioning

confidence: 99%

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components

Pirbadian

Barchinger

Leung

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

573

469

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Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution. R eduction-oxidation (redox) reactions and electron transport are essential to the energy conversion pathways of living cells (1). Respiratory organisms generate ATP molecules-life's universal energy currency-by harnessing the free energy of electron transport from electron donors (fuels) to electron acceptors (oxidants) through biological redox chains. In contrast to most eukaryotes, which are limited to relatively few carbon compounds as electron donors and oxygen as the predominant electron acceptor, prokaryotes have evolved into versatile energy scavengers. Microbes can wield an astounding number of metabolic pathways to extract energy from diverse organic and inorganic electron donors and acceptors, which has significant consequences for global biogeochemical cycles (2-4).

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“…This appears to rule out a mechanism of immobilization due to the binding of EpsE to the flagellar motor (see also Supplementary Fig. S6), which occurs in B. subtilis biofilms (Blair et al, 2008). Interestingly, front-line cells at the extreme edge of tips were also normally static, but if individuals were reincorporated into the interior as the tip advanced, they immediately became mobile (Supplementary Movie Files S1-S3).…”

Section: Non-swarmers Apparently Physically Immobilizedmentioning

confidence: 97%

Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers

et al. 2011

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Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers The non-domesticated Bacillus subtilis strain 3610 displays, over a wide range of humidity, hyperbranched, dendritic, swarming-like migration on a minimal agar medium. At high (70 %) humidity, the laboratory strain 168 sfp + (producing surfactin) behaves very similarly, although this strain carries a frameshift mutation in swrA, which another group has shown under their conditions (which include low humidity) is essential for swarming. We reconcile these different results by demonstrating that, while swrA is essential for dendritic migration at low humidity (30-40 %), it is dispensable at high humidity. Dendritic migration (flagella-and surfactin-dependent) of strains 168 sfp + swrA and 3610 involves elongation of dendrites for several hours as a monolayer of cells in a thin fluid film. This enabled us to determine in situ the spatiotemporal pattern of expression of some key players in migration as dendrites develop, using gfp transcriptional fusions for hag (encoding flagellin), comA (regulation of surfactin synthesis) as well as eps (exopolysaccharide synthesis). Quantitative (singlecell) analysis of hag expression in situ revealed three spatially separated subpopulations or cell types: (i) networks of chains arising early in the mother colony (MC), expressing eps but not hag; (ii) largely immobile cells in dendrite stems expressing intermediate levels of hag; and (iii) a subpopulation of cells with several distinctive features, including very low comA expression but hyper-expression of hag (and flagella). These specialized cells emerge from the MC to spearhead the terminal 1 mm of dendrite tips as swirling and streaming packs, a major characteristic of swarming migration. We discuss a model for this swarming process, emphasizing the importance of population density and of the complementary roles of packs of swarmers driving dendrite extension, while non-mobile cells in the stems extend dendrites by multiplication. INTRODUCTIONThe formation of bacterial communities, such as Bacillus subtilis colonies, was shown recently, while this work was in progress, to occur through a developmental-like program, associated with the presence of different cell types (see Vlamakis et al., 2008;Lemon et al., 2008;Ló pez & Kolter, 2010). Another form of community swarming migration over a surface, apparently occurring in thin fluid films, requires flagella, a surfactant and the production of specialized swarmer cells ( , 2008). Two types of swarming-like migration by B. subtilis have been described. On Luria broth (LB) agar at low humidity and 37 u C (primarily analysed with nondomesticated strains such as 3610), migration proceeds as an expanding confluent field, headed by small groups of bacteria, apparently with little change in individual size (Kearns & Losick, 2003;Julkowska et al., 2004). In contrast, we have described the...

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A Molecular Clutch Disables Flagella in the Bacillus subtilis Biofilm

Cited by 298 publications

References 24 publications

A molecular brake, not a clutch, stops the Rhodobacter sphaeroides flagellar motor

A molecular brake, not a clutch, stops the Rhodobacter sphaeroides flagellar motor

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components

Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers

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