Why flagella cannot be seen

The microbe uses this to determine which way is up and that helps it to find nutrients or adjust its depth in an aquatic environment. Other animals have magnetosomes; birds, dolphins, tuna, green turtles. In these cases they are used for navigation on long migrations. Chemotaxis is accomplished by sensing the environment and adjusting the rotation of the flagella in response to stimuli. Before we get into this, I will point out that most of this work has been done on E.

Chemotaxis can behave differently in other microbes. Flagella can rotate clockwise or counterclockwise. When flagella rotate counterclockwise this creates a force pushing on the bacteria.

In the case of E. This causes the bacteria to move in a straight line, called a run. When flagella rotate clockwise, they all pull on the microbe. With all these forces pulling in different directions, it causes the bacteria to tumble or twiddle.

When the twiddling is over, the bacteria will start out a new run in a completely random direction. In plain medium containing no attractant or repellent, the length of runs is random and the bacteria move about the solution aimlessly.

Shown here is an animation of a microbe moving in a neutral environment with no attractant or repellent. When cells are put in an environment containing an attractant such as glucose, they will move toward the source of the attractant. The microbes are sensing the change in concentration of the attractant, the concentration gradient , as they move through solution. If they are moving up the gradient to higher attractant concentrations, the length of the run will increase.

If they are moving down the gradient, the length of the run will be much shorter. In this way, the bacteria eventually moves to the source of the attractant. Shown here is an animation of a microbe moving in an environment containing a gradient of attractant. The behavior here is exactly the opposite of an attractant. When the compound is nasty, such as HCl an acid , the microbe will shorten runs that go up the gradient and lengthen those that take it away from the repellent.

The basal body complex harbors a flagellum-specific protein export machine 2. This flagellar-specific type-III secretion system exports most extra-cytoplasmic building blocks of the flagellum in a proton motive force PMF dependent manner 3 , 4. In Salmonella enterica , expression of flagellar genes is temporally coupled to the assembly state of the flagellum and can be ordered into a transcriptional hierarchy of three promoter classes 9 , Gene products expressed from Class 2 promoters include the components of the HBB complex, as well as regulatory proteins, e.

The completion of the HBB complex results in a switch in secretion-substrate specificity within the type-III secretion apparatus from secretion of early HBB-type substrates to the secretion of late filament-type substrates. Class 3 gene products are needed for completion of the flagellum e. It is presumed that shearing of flagellar filaments occurs in nature, however it is not clear if a sheared flagellum can re-grow A sheared flagellum would need to re-assemble the filament cap structure, as the original cap would have been lost by the shearing event.

In Salmonella enterica , the filament cap gene fliD is transcribed from both Class 2 and Class 3 promoters After the switch in substrate secretion specificity, FliD is secreted simultaneously with FlgM allowing for an efficient transition to filament assembly.

Thus, the FliD cap does not compete with flagellin for secretion prior to initiation of flagellin gene expression. FliD is likely expressed from its Class 3 promoter in the case of shearing events, which allows the formation of a new cap on the tip of the broken filament and thus re-growth of a sheared filament. In order to determine if flagellar filaments can re-grow, Rosu and Hughes analyzed the dynamics of Class 3 gene expression after flagellar shearing in Salmonella enterica FlgM is constantly secreted during flagellar growth.

The rate of flagellar substrate export decreases with the length of the filament structure 6 , Thus, shearing of a filament might result in a sudden increase in the rate of FlgM secretion and a subsequent burst of Class 3 gene expression. However, the levels of intracellular FlgM and Class 3 gene expression remained unchanged after flagellar shearing In a recent study, Turner and colleagues used fluorescent bi-color labeling of flagellar filaments to measure filament growth in Escherichia coli in a population-approach Differential fluorescent labeling of flagellar filaments allowed the authors to distinguish the growth of new filament segments from previously grown parts of the same filament.

Turner et al. The authors concluded that broken flagellar filaments of E. However, a caveat of their experiment was the inability to distinguish re-growth of sheared filaments from continued growth of nascent, short filaments that were not broken.

In the present study, we used three independent techniques to unambiguously determine whether individual flagellar filaments re-grow after being damaged.

We demonstrate using 3-color differential fluorescent labeling of flagellar filaments that flagellar filaments are indeed able to re-grow after breakage by mechanical shearing forces.

We further visualized, for the first time, the formation of new cap structures on broken filaments using differential electron microscopy. Finally, using again fluorescent labeling, we monitored the growth of individual filaments that were broken one by one with ultrashort laser pulses. Filaments broken with this method were not observed to re-grow, in contrast to the mechanically sheared ones.

Thus, we conclude that re-growth of flagellar filaments depends on the method of breakage. The differential fluorescent dual-color labeling of flagellar filaments pioneered by Turner and colleagues 15 was modified by adding a third labeling step 6 , which allowed us to unequivocally determine if a broken filament re-grew. Residue T in the variable loop of the FliC flagellin of Salmonella enterica was chosen for cysteine substitution because it is accessible for external labeling Figure S1A.

After a pulse induction of the expression of flhDC , incubation of strain EM was resumed in the absence of inducer to prevent formation of another round of basal-body complexes, which facilitated length measurements of the filament segments. Subsequently, expression of fliC TC was induced from the chromosomal P araBAD promoter in the presence of the first fluorophore coupled to a cysteine-specific maleimide moiety. This allowed for the simultaneous initiation of flagellar filament assembly and labeling of the first filament segment F1.

The filament segment F2 grew to an average length of approximately 3. The control sample was manipulated the same way, except that filaments were not mechanically sheared. The 3-color labeling allowed us to determine if a filament stopped growing or had been broken by mechanical shearing forces.

In case of the control sample, we observed only flagella labeled with a pattern of F2-F3 or F1-F2-F3 segments, which demonstrated that none of the flagella stopped growing or broke during the duration of the experiment Fig. Broken flagella re-grow after mechanical damage. A Top: F1, F2 and F3 filament fragment lengths of the control sample after 2-color labeling left panel and 3-color labeling right panels. Bottom: Representative fluorescent microscopy images. B Top: F1, F2 and F3 filament fragment lengths of the shearing sample after 2-color labeling pre-shearing, left panel and 3-color labeling post-shearing, right panels.

We considered only filaments that displayed a labeling pattern of F1-F3 i. This demonstrated that re-growth could occur after shearing Fig. For the remaining 38 filaments, which retained a F2-F3 or F1-F2-F3 pattern, the pattern of filament segments does not unequivocally demonstrate that they were broken.

However, the short length of the F2 fragment average F2 length of 0. Broken flagellar filaments have lost their original filament cap and would need the ability to re-assembly a new capping structure before they are able to resume growth. We thus tested whether we could observe the formation of a new filament cap on top of previously sheared flagella. A hemagglutinin HA epitope tag was inserted in the cap plate accessible area of the filament cap FliD to facilitate visualization by immuno-gold electron microscopy 16 , Strain TH harbored i the flagellar master regulatory operon under control of an AnTc inducible promoter P tetA - flhDC for synchronized production of flagellar basal bodies, ii the flagellin fliC TC cysteine substitution at residue T in the variable loop of the FliC flagellin expressed from the chromosomal fliC locus for labeling of sheared filament segments, and iii the HA-epitope tagged fliD construct under control of an arabinose-inducible promoter P araBAD - fliD ::HA in addition to the native fliD gene.

The inducible HA-epitope tagged FliD construct allowed us to express FliD-HA at later stages of filament assembly and thus visualize formation of newly formed cap structures on the tip of broken filaments.

After a pulsed induction of flhDC expression, incubation of strain TH was resumed in the absence of AnTc inducer to prevent formation of another round of basal-body gene expression. Gold-labeling of the initially assembled filament allowed us to identify basal filament segments that were made prior to any shearing event. Excess gold maleimide was removed by mild centrifugation and the flagellar filaments were mechanically sheared as described above.

Prior to mechanically shearing the flagella, we induced expression of HA-epitope tagged FliD by addition of arabinose in order to visualize newly formed cap structures. Since wild type fliD was constitutively expressed from its native locus during the experiment, both HA-epitope tagged FliD and wild type FliD were secreted simultaneously, and this reduced the probability to observe cap structures formed only by HA-epitope tagged FliD.

The overproduction of arabinose-induced fliD:: HA increased the probability of detecting the assembly of a new HA-epitope tagged FliD cap on the tip of a broken filament, however, the low frequency prevented an adequate quantitative analysis of the probability of re-growth and formation of new capping structures. The filament structures were stained with methylcellulose-uranyl acetate and visualized by electron microscopy.

Formation of a new filament cap on re-grown flagellar filaments. Flagellar filaments were gold-labeled before mechanically shearing the flagella using viscous shearing forces.

As expected, we observed shorter gold-labeled basal filament segments for flagella that were mechanically sheared compared to the gold-labeled basal filament segments of not-sheared control samples Fig.

We considered flagellar filaments that displayed short, gold-labeled basal filament segments followed by an unlabeled filament segments as flagella that had successfully been sheared.

Importantly, we observed HA-epitope tagged FliD caps on the tip of such previously sheared flagellar filaments Fig. Thus, the detection of HA-epitope tagged FliD on the tip of previously broken and re-grown filaments unequivocally demonstrates that a new filament cap can reassemble to allow re-growth of broken flagella.

An alternate method to break filaments was then used in order to test the possibility that re-growth of flagellar filaments depends on how they were broken. Ultrashort laser pulses are frequently used to manipulate biological tissues 18 , 19 , 20 , Physical damage to the material is not thermal, but rather induced by the emission of a shock wave driven by the rapid expansion of a laser-induced plasma a so-called Coulomb explosion 22 , In other words, the use of ultrashort pulses implies that very little energy is deposited essentially no heat and that the material near its focal point is damaged by the creation of a very localized shock wave.

That shock wave was used to break individual flagellar filaments placed in the vicinity of the laser beam. In order to break individual flagellar filaments and observe their re-growth, we needed an experimental setup that allowed for the unambiguous identification of individual filaments on a microscope slide over multiple hours. That was greatly simplified by using a bacterial strain for which the majority of cells possessed on average a single filament.

However, it was recently reported that the requirement for fliO could be bypassed by mutations in fliP Only cells that were firmly attached to the coverslip, and that displayed a single flagellum were selected for laser damage of the filament.

In order to ensure that the observed cell was alive and healthy, we considered only cells with rotating filaments. In addition, we selected filaments that were not only rotating on their axis, but also slowly gyrating i. Indeed, initial trials showed that if the filament was not gyrating, the laser pulses which broke the filament frequently stopped the rotation of the motor.

It is not exactly clear why that was the case, but presumably non-gyrating filaments have a much stronger tendency to stick to the cell body or the coverslip when shortened. The ideal candidate was therefore a rotating filament that was also gyrating in a somewhat uniform circular trajectory. A successful filament breakage was clearly identified by the acceleration of the filament gyration, and the broken filament fragment was often seen diffusing away. To establish that the laser did not damage the flagellar motor or compromise the cell membrane, we made sure that the broken filament was still rotating.

Figure 3 shows the same bacterium before panel A and after panel B its filament was broken. The full movie is available in the Supplemental Material online Movie S1.

Flagellar filaments broken using ultrashort laser pulses do not re-grow. The cell body is barely visible highlighted with white dotted line and the filament shows up large and fuzzy because it is rotating much faster than the image acquisition rate. The white arrow points to the broken filament segment drifting away and out of focus.

In simple staining , a single dye is used to emphasize particular structures in the specimen. A simple stain will generally make all of the organisms in a sample appear to be the same color, even if the sample contains more than one type of organism.

In contrast, differential staining distinguishes organisms based on their interactions with multiple stains. In other words, two organisms in a differentially stained sample may appear to be different colors.

Differential staining techniques commonly used in clinical settings include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining. Table 3 provides more detail on these differential staining techniques. The Gram stain procedure is a differential staining procedure that involves multiple steps.

It was developed by Danish microbiologist Hans Christian Gram in as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques.

The steps of the Gram stain procedure are listed below and illustrated in Table 1. Gram-staining is a differential staining technique that uses a primary stain and a secondary counterstain to distinguish between gram-positive and gram-negative bacteria.

Step 2: Iodine. Cells remain purple or blue. Step 3: Alcohol. Step 4: Safranin. Gram-negative cells appear pink or red. Figure 3. In this specimen, the gram-positive bacterium Staphylococcus aureus retains crystal violet dye even after the decolorizing agent is added. Gram-negative Escherichia coli, the most common Gram stain quality-control bacterium, is decolorized, and is only visible after the addition of the pink counterstain safranin.

The purple, crystal-violet stained cells are referred to as gram-positive cells, while the red, safranin-dyed cells are gram-negative Figure 3. However, there are several important considerations in interpreting the results of a Gram stain. First, older bacterial cells may have damage to their cell walls that causes them to appear gram-negative even if the species is gram-positive.

Thus, it is best to use fresh bacterial cultures for Gram staining. Second, errors such as leaving on decolorizer too long can affect the results. In some cases, most cells will appear gram-positive while a few appear gram-negative as in Figure 3. This suggests damage to the individual cells or that decolorizer was left on for too long; the cells should still be classified as gram-positive if they are all the same species rather than a mixed culture.

Besides their differing interactions with dyes and decolorizing agents, the chemical differences between gram-positive and gram-negative cells have other implications with clinical relevance. For example, Gram staining can help clinicians classify bacterial pathogens in a sample into categories associated with specific properties. Gram-negative bacteria tend to be more resistant to certain antibiotics than gram-positive bacteria. We will discuss this and other applications of Gram staining in more detail in later chapters.

Figure 4. However, more information is needed to make a conclusive diagnosis. The technician decides to make a Gram stain of the specimen. This technique is commonly used as an early step in identifying pathogenic bacteria. After completing the Gram stain procedure , the technician views the slide under the brightfield microscope and sees purple, grape-like clusters of spherical cells Figure 4.

Acid-fast staining is another commonly used, differential staining technique that can be an important diagnostic tool. An acid-fast stain is able to differentiate two types of gram-positive cells: those that have waxy mycolic acids in their cell walls, and those that do not.

Two different methods for acid-fast staining are the Ziehl-Neelsen technique and the Kinyoun technique. Both use carbolfuchsin as the primary stain. The waxy, acid-fast cells retain the carbolfuchsin even after a decolorizing agent an acid-alcohol solution is applied.

A secondary counterstain, methylene blue, is then applied, which renders non—acid-fast cells blue. The fundamental difference between the two carbolfuchsin-based methods is whether heat is used during the primary staining process. The Ziehl-Neelsen method uses heat to infuse the carbolfuchsin into the acid-fast cells, whereas the Kinyoun method does not use heat.

Both techniques are important diagnostic tools because a number of specific diseases are caused by acid-fast bacteria AFB. If AFB are present in a tissue sample, their red or pink color can be seen clearly against the blue background of the surrounding tissue cells Figure 5.

Figure 5. Ziehl-Neelsen staining has rendered these Mycobacterium tuberculosis cells red and the surrounding growth indicator medium blue. Mycobacterium tuberculosis , the bacterium that causes tuberculosis , can be detected in specimens based on the presence of acid-fast bacilli.

If acid-fast bacteria are confirmed, they are generally cultured to make a positive identification. Variations of this approach can be used as a first step in determining whether M. An alternative approach for determining the presence of M. In this technique, fluorochrome-labeled antibodies bind to M. A Rotne-Prager matrix was used for calculating the cross-mobilities and cross-hydrodynamics Dhont, The swimming dynamics of the model cell at low Reynold number was calculated by solving the translational and rotational Stokes equations of motion for the cell body, flagellar beads and the bonds between them.

A second-order Runge-Kutta algorithm Sewell, ; Press et al. An exemplary track of a fast reorientation event is given in Figure 1—figure supplement 3. The algorithm for 3D track event detection was validated using Langevin-simulations of active Brownian particles with defined mean reorientation angles, velocities, mean run times and mean run lengths, as described in Figure 1—figure supplement 1.

Two parameter sets have been simulated and used for validation. The evaluation shows good agreement between input and output parameters. The swim track parameters that have been determined from 3D tracks of MC-1 cells in the capillary experiment are presented in Figure 1—figure supplement 2. While the contribution of the small helix to the actual swimming path provides another factor of 1. In these simulations the motor torque of one flagellum Tm2 is 0.

We observed double-helical trajectory with this motor asymmetry as well but with considerably smaller size compared to the experiment and our proposed pusher-puller model. The size of helix and swimming features are presented in Table 1 , as well as experimental values for comparison. Also, the simulated velocities did not fit the experimental values for any of the tested scenarios. We performed simulations with asymmetry in flagella direction. In swimming trajectories, a very weak second helix with diameters of about an order of magnitude smaller than our pusher-puller model was observed.

In these simulations, the more tilted flagella bundle is only tilted close to the cell surface, but due to flexibility, the tilt is not persistence along the length of the flagella bundle and the two bundles approach each other at their end parts. While the pitch of the simulation result, presented in the main text, is off by a factor of 2, the diameter and period time of helical trajectories are in good agreement with the experiments.

The simulations show that the features of helical trajectories strongly depend on the flagellar opening angle see Table 2 such that increasing the flagellar opening angle decreases the pitch and diameter of the large helix.

Simulated effective velocities are smaller than the tracked velocities by factor of 2, since the pitch is decreased by the same factor while the period times are comparable. Since the simulations with constant flagella length did not lead to a satisfying match in pitches, further simulations for different combinations of opening angles and flagellum lengths were carried out. The resulted diameter, pitch and velocity are compared with experimental values in Figure 5—figure supplement 2.

However, none of them satisfy all the parameters simultaneously. The best match between helix diameter, pitch and speed is described in the main text. However, we clarify here that other tuning parameters for example flagellum length and diameter, flagella opening angle, the motor torque in the model cannot be precisely determined from TEM or optical microscopy images of MC Therefore, it is computationally very time consuming to test different values for all these tuning parameters to find an accurate quantitative match for all parameters between experiment and simulation.

We further investigated several motor torques. However, we can extrapolate the swimming behavior of our model MC-1 at higher motor torques by looking at its trend for lower values of motor torque. We tried motor torques of 2, 2. The top row represents the result discussed in the main text. The overall pitch increases with increasing motor torque. It can be concluded that the motor torque can be one of the parameters that may help matching between experiment and simulation, although the definitive match was not reached in this study.

Three possible transient flagella configurations for reorientation events were tested in the simulations CCW meaning counter-clockwise and CW meaning clockwise. The MC-1 cell shape is given in Figure 3A in the main text, where seven individual flagella were identified, emerging from a sunken pit, as already described in Bazylinski et al.

The individual bundles of the close relative MO-1 Ruan et al. Additionally, 24 gap-filling, presumably friction-reducing microfibrils were found in Ruan et al. We assumed a similar flagella arrangement in this study, leading to a cooperative torque generation of the individual flagella in one sheath of an MC-1 cell.

Over tracks are extracted and investigated for each magnetic field. For more certainty, swimming trajectories are investigated both in oxic and anoxic regions. In high magnetic field, over 80 trajectories can be detected illustrating the hyper-helix, while in Earth magnetic field only a few trajectories with semi-hyper-helix can be observed.

A typical MC-1 trajectory with the mentioned hyper-helix resulted from simulation and experiment is shown in Figure 4D. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses. This paper reports on the discovery of an unusual and fascinating form of motility in a magnetotactic bacterium.

Through a combination of experiment and theory the authors have found that the two flagellar bundles work in opposite ways — one pushing and one pulling the cells through their fluid environment in double-helical paths.

The very fast motility and very rapid trajectories changes appear to arise from these features. This work will surely be of interest not only to those interested in the biology of motility, but also physical scientists interested in the fluid dynamics of locomotion. Thank you for sending your article entitled "High-speed motility originates from cooperatively pushing and pulling flagella bundles in bilophotrichous bacteria" for peer review at eLife.

Your article is being evaluated by three peer reviewers, and the evaluation is being overseen by a Reviewing Editor and Detlef Weigel as the Senior Editor. The essence of the criticisms concerns the interpretation of the experimental observations and the possibility of achieving better imaging of the flagella and the analysis of the computational results. In both cases further details are needed, and in the former it appears that additional imaging would be in order.

However, I am not completely convinced by the authors' evidence. As detailed below, while the story is quite plausible, the experiments seem to not have key evidence which I would have expected to have easily been observed if the scenario were true, and the numerics are not performed or perhaps just not described in a way that convincingly rules out other hypotheses.

This identification is not obvious to me, and it is quite puzzling why the authors do not actually image the flagellar bundles.

If the bundles were indeed extended from the body in front and behind the cell, I would expect that they could be visualized which would definitively prove their proposed configuration.

In Son, Guasto and Stocker, , sheathed flagella are quite readily imaged. Note that fps is more than fast enough to resolve the larger helical motion with period 72 ms. It is unclear if the authors attempted this. If the authors tried and were not able to see a leading and lagging bundle, I would take that as evidence against their claim.

The smaller helix has in the past been attributed to the rotation of the bundle or flagellar helix, see Keller and Rubinow, The larger helix arises anytime there is non-axisymmetric propulsion, see Hyon, Powers, Stocker, Fu and has been observed in many species, albeit with varying sizes of the larger helix. As discussed in point 3 below, an important implication of this is that the bar for numerical simulations cannot be simply qualitative as in "straight" vs.

First, as mentioned above, the quantitative details of the helical trajectory are important, but these are not well-matched. The authors say that their trajectories match the experimental helical diameter and period of the trajectories but not the pitch. The results are strongly dependent on the angle between the bundles, which is used as a fitting parameter. They say that they believe that the pitch could be matched eventually by fitting the opening angle and flagella length but do not actually do it.

Second, changing directions of bundles is also likely able to produce many different types of helical trajectories with varying pitch and radii. These have not been eliminated as possibilities. Third, the fast reorientation in the simulations is interesting, but again, it is not ruled out whether other configurations could also yield similarly fast reorientations. Fourth, the speed of the swimming is used as supporting evidence, but this is achieved by increasing the torque on the motor to a value somewhat higher than some report for E.

If one is allowed to adjust the motor torque, then any speed can be reached for any configuration. The manuscript reports results of a detailed experimental and numerical study of the swimming motion of magnetotactic cocci, bilophotrichous bacteria with flagellar bundles at both poles. The experiments show that bacteria swim along a double helical path, with a very high swim speed and short reorient time compared to other bacteria. Hydrodynamic modelling demonstrates that this is due to a pushing and a pulling bundle.

Experimental and numerical results are in good semi-quantitative agreement. I think this is a very nice investigation, which carefully elucidates the complex swimming motion of a bilophotrichous bacterium. Experiment and hydrodynamic simulation complement each other very well to characterize the geometry of the flagellar organization and swim pattern.

Thus, I strongly support publication of this manuscript in eLife. I have only a few comments, which the authors should consider before publication:. I found this pretty confusing, until I saw Figure S1. I recommend clarifying this point early in the main text. This would explain the particular value of the reorientation angle.

This seems to be somewhat similar to the behavior seen in simulations of the early stages of swimming and bundle formation of peritrichous bacteria, when the flagella are initially pointing in arbitrary directions, see, J.

Hu et al. However, the bundle rotation direction has to change back. Please clarify.