Why do flagella form a bundle only when they rotate counterclockwise during chemotaxis?

During Chemotaxis in bacteria with flagella, the flagellar rotation dictates how the cell moves. If the flagella rotate counterclockwise, then they form a bundle at one end of the cell (---O) and propel it forward in a coordinated fashion. However, if the flagella rotate clockwise, then each flagella acts independently to push the cell in many different directions.

My question is, why doesn't clockwise flagellar rotation simply cause the bundle to form on the opposite end of the cell? (O---)

In principle this can happen, but you have to read up on flagellar dynamics and polymorphic transitions (Real-Time Imaging of Fluorescent Flagellar Filaments, Turner et al. 2000).

From Howard Bergs lab page:

Runs can occur with filaments of any polymorphic form; although, the normal form predominates. For a cell to tumble, not every filament needs to change its direction of rotation. Different filaments can change directions at different times, or a tumble can result from the change in direction of only one.

If the whole bundle comes apart it can reform on either end of the cell body because E. coli has a random flagellation pattern (peritrichous) and thus no preferred direction of propagation of the cell body.

Also have look here for some nice movies from the Berg lab.

A statistical physics view of swarming bacteria

Bacterial swarming is a collective mode of motion in which cells migrate rapidly over surfaces, forming dynamic patterns of whirls and jets. This review presents a physical point of view of swarming bacteria, with an emphasis on the statistical properties of the swarm dynamics as observed in experiments. The basic physical principles underlying the swarm and their relation to contemporary theories of collective motion and active matter are reviewed and discussed in the context of the biological properties of swarming cells. We suggest a paradigm according to which bacteria have optimized some of their physical properties as a strategy for rapid surface translocation. In other words, cells take advantage of favorable physics, enabling efficient expansion that enhances survival under harsh conditions.


1. Pili - straight hairlike appendages they are usually short all gram negative bacteria have pili function is to attach bacteria to other bacteria, other cells, or other surfaces (not for locomotion):

a. sex pili allow one bacterial cell to adhere to another (cells can actually exchange genetic material through the pili - this is the closest bacteria get to sexual reproduction!) called conjugation.

b. other types of pili attach bacteria to plant or animal cells to maintain themselves in a favorable environment if pili have been lost (maybe due to a mutation) in disease-causing bacteria, the bacteria will not be able to establish an infection.

2. Flagella (singular – flagellum) - long, thin structures that extend outward from the surface of the envelope function is locomotion - bacteria with flagella are motile flagella rotate to propel the bacterium. Bacteria can have 1, 2, or many flagella (ex. of a bacteria with many flagella – Salmonella).

3. Axial Filaments - bundles of flagella which wrap around the cell body between the cell wall and the outer membrane together they form a helical bulge that moves like a corkscrew as the entrapped flagella turn & propel the cell found only in one type of bacteria called the spirochetes this unique form of movement is well suited to the viscous environment (mud & mucous) where the bacteria is generally found. Ex. of bacteria with a.f. – Treponema (causes syphilis) and Borrelia (causes Lyme disease).

B. Cell Envelope (layers from outside to inside) (BE ABLE TO DIAGRAM!)

1. Glycocalyx - found in most bacteria slimy or gummy substance that becomes the outermost layer of the cell envelope a thick glycocalyx is often called a capsule a thin glycocalyx is often called a slime layer functions:

a. protection from drying out

b. helps a cell adhere to a surface where conditions are favorable for growth

c. provide protection against phagocytosis (engulfment & destruction by cells such as white blood cells) - a slippery glycocalyx makes it difficult for the phagocyte to grab hold of the bacterium.

2. Outer Membrane - primarily found in gram negative bacteria (ex. E. coli, Salmonella, Shigella, Pseudomonas, Proteus, Neisseria gonorrhoeae) composed of a bilayer membrane the inner layer is composed of phospholipids the outer layer is composed of lipopolysaccharides (LPS’s), a compound that's not found in any other living organism! part of the LPS is hydrophobic, part is hydrophilic most molecules are transported across the outer membrane and into the cell through special proteins called porins these porins create small pores or channels in the outer membrane that allow molecules to diffuse in function of the outer membrane is mainly protection - because of the outer membrane, gram negative bacteria are generally more resistant than gram positive bacteria to many toxic compounds, including antibiotics (antibiotics are too large to diffuse through the porins).

More about LPS’s – These compounds are endotoxins and are only released when the bacteria die and their cell walls are broken down. Endotoxins cause fever and dilate blood vessels (drop in blood pressure results). Killing the bacteria may increase the concentrations of this toxin!

3. The Cell Wall - The structure described below is found in all eubacteria except the mycoplasmas (these bacteria lack a cell wall) in archaeobacteria, the cell walls are composed of a different type of peptidoglycan or protein & some do not have cell walls. In gram negative bacteria, the cell wall lies just inside the periplasm in gram positive bacteria, it lies just inside the glycocalyx, if one exists.

a. Structure & Composition of Cell Wall in Eubacteria

1.) The chief component is peptidoglycan.

2.) Peptidoglycan is composed of long chains of polysaccharides (glycan) cross-linked by short proteins (peptides).

3.) When linked together these chains create the single rigid mesh-like molecule that forms the bacterial cell wall (resembles a chain link fence!)

4.) A major difference between G(+) & G(-) bacterial cell walls:

a.) G(-): peptidoglycan mesh is only one layer thick.

b.) G(+): peptidoglycan wall is many layers thick.

b. Cell Wall Function – In many cases, the cell wall is very porous and does not regulate the transport of substances into the cell. Two major functions of the cell wall are maintaining shape and withstanding turgor pressure. Both are discussed below.

1.) Cell Shape - one fxn. of the cell wall is to confer shape on the bacterium most bacteria fall into one of these general groups. However, some bacteria have irregular shapes. Even bacteria of the same kind or within the same culture sometimes vary in size and shape (especially in aging cultures).

a.) cocci (singular - coccus) - spherical

b.) bacilli (singular - bacillus) - rod-shaped

c.) spirilli (singular - spirillum) - spiral-shaped

In addition to these characteristic cell shapes, cells can also be found in distinctive groups of cells: pairs, chains, tetrads (cubes), grape-like clusters, etc.

2.) Withstanding Turgor pressure – A cell's turgor pressure is the internal pressure from its contents. Ordinarily, a bacterium is in a hypotonic solution (a more dilute solution that has less solute and more water than the inside of the bacterium) and water tries to move from a high water concentration to a low water concentration that is, water tries to move inside the bacterium (see tonicity under osmosis later in the handout). Without the cell wall, the water would continue to more inside the cell, and the cell would lyse or burst the cell wall withstands turgor pressure, so that the cell does not lyse.

Action of some antibiotics (ex. penicillin) - Bacteria produce enzymes that reseal breaks in the peptidoglycan cell wall that occur during normal growth and division penicillin binds to these enzymes, inactivating the enzymes so that the breaks cannot be resealed. The bacteria then lyse.

Lysozyme, an enzyme found in tears, digests (breaks down) peptidoglycan.

c. Mycoplasmas - group of bacteria that lack a cell wall they avoid lysis from turgor pressure by maintaining a nearly equal pressure between their cytoplasm and their external environment by actively pumping sodium ions out of the cell additionally, their cell membranes are strengthened because they contain cholesterol, a lipid found in eukaryotic cell membranes.

4. Periplasm - used to be called a space, because of the way it looked in electron micrographs found between the cell membrane and the peptidoglycan cell wall therefore, only found in gram negative cells composed of a gelatinous material containing proteins one function of these proteins is that break down certain nutrients into smaller molecules that can pass through the cell membrane.

5. Plasma or Cell Membrane - membrane that encloses the cytoplasm of any cell major function is to contain the cytoplasm and to transport and regulate what comes in and what goes out of the cell. Many prokaryotic cell membranes are similar to eukaryotic cell membranes. Its structure is referred to as the Fluid Mosaic Model, because the structure behaves more like a fluid than a solid. Contains:

Membrane Lipids: (composed primarily of phospholipid molecules)

a.) phospholipid bilayer (hydrophobic fatty acid tails & hydrophilic phosphate heads review chemistry handout on phospholipids)

Membrane Proteins: (proteins float in the fluid lipid bilayer)

a.) Integral proteins - inserted in the bilayer mainly involved in transport.

1.) carrier proteins - bind to specific substances & transport them across the cell membrane.

2.) channel proteins - proteins with a channel through which small, water soluble substances move across the cell membrane.

b.) Peripheral proteins - usually attached to membrane surface some are enzymes some are involved in the electron transport chain and/or photosynthesis (we’ll talk about these processes in the metabolism chapter) others are involved in the changes in cell shape that occur during cell division.

Note: Archaeobacteria Cell Membranes - there are different kinds of bonds in the phospholipid molecules that link the lipids (tails) to the glycerol molecule (head) these bonds are stronger and may help these bacteria survive extreme temperature and pH.

Cell Membrane Invaginations - the cell membrane sometimes invaginates or folds back on itself, forming structures that extend into the cytoplasm since prokaryotic cells lack organelles, these invaginations provide increased surface area for peripheral proteins (enzymes) to catalyze chemical reactions.

C. Cytoplasm - matrix composed primarily of water (90%) & proteins. Contains the following:

1. Nucleoid - or nuclear region is a mass of DNA well defined, although it is not surrounded by a membrane most of a bacterium's DNA is arranged in a single circular molecule called a chromosome some bacteria also contains smaller circular DNA molecules called plasmids (to be discussed later).

2. Ribosomes - site of protein synthesis prokaryotic ribosomes are smaller than eukaryotic ribosomes. Antibiotics such as tetracycline, erythromycin, and streptomycin can specifically target bacterial ribosomes & not harm the host's eukaryotic ribosomes.

3. Endospores - extremely hardy, resting (non-growing) structures that some bacteria, principally G(+), produce through the process of sporulation when nutrients are exhausted when favorable conditions return, endospores germinate to produce new vegetative cells, which grow & reproduce they are able to withstand harsh environmental conditions because they contain so little water and high concentrations of calcium and dipicolinic acid when favorable conditions return, the spore germinates into a new vegetative cell.

Some of endospore-producing bacteria are pathogenic to humans. Ex. Clostridium tetani causes tetanus (other species of this genus cause botulism and gas gangrene). Bacillus is another genus of bacteria that forms spores. We will learn how to stain bacteria so you may observe these spores.


Eukaryotic cilia and flagella are highly conserved and ubiquitous organelles present in most animals, as well as many lower plants and eukaryotic protozoa. In the vertebrate lineage, the eukaryotic flagellum is universally employed to propel the male gamete. In the mammalian branch of the vertebrates, the sperm tail is always a modified flagellum with some characteristic accessory features that are added onto the basic 9 + 2 axoneme of microtubules found in most cilia and flagella.

Figure 1 shows transmission electron micrographs (TEMs) of a mouse sperm flagellum in cross section at several positions along the length of the flagellum. The central axoneme is visible with its nine outer doublets surrounding a pair of single central microtubules (central pair or CP). Each of the outer microtubule doublets bears two rows of projections called the inner row and outer row of dynein arms. These arms are composed of the dynein motor proteins that power motility. The dynein arms are the source of the motive force that bends the flagellum. The dynein heavy chains (DHC) of the arms form bridges between the outer doublets and undergo a Mg-ATP driven power stroke that generates a sliding (or shearing) action between the doublets.

Ultrastructure of a mammalian sperm flagellum.

TEM of mouse sperm flagella. A series of cross sections are displayed that show the axoneme and periaxonemal elements of a mouse sperm flagellum at progressively more distal positions along the flagellum from panel A to E. The sections are not from the same flagellum but have been aligned to show comparative features in the same orientation. MS, FS, ODF, doublet microtubule (MT), LC of the FS, RS, CP, inner dynein arm, and outer dynein arm are labeled. ODFs 1, 5, and 6 are labeled following the standard numbering convention. Bar = 200 nm.

As is apparent in Figure 1, the centrally located 9 + 2 axoneme of microtubules has some rather substantial additional structures surrounding it. These structures are common to the sperm tails of mammalian species and often dwarf the central axoneme. The first additional set of structures is nine large fibers paired to each of the doublets. These are called the outer dense fibers (ODFs). Unlike the doublets, they are not composed of tubulin, but are intermediate filament-like structures that are made of keratin-like material (Olson, 1979 Brohmann et al., 1997 Olson and Sammons, 1980 Peterson, 1982 Kierszenbaum, 2002 Rivkin et al., 2008 ). The ODFs are not uniform in length. Those associated with doublets number 1, 5, and 6 are longest and extend ∼¾ of the length of the flagellum, while those of doublets 3 and 8 are shortest and end at the junction of the midpiece with the principal piece (Lindemann and Gibbons, 1975 Serres et al., 1983 Lindemann et al., 1992 ).

Surrounding the ODFs in Figure 1A is a thick layer of mitochondria that form a sheath around the flagellum just under the outer membrane of the cell. In bull sperm, this mitochondrial sheath (MS) covers the first 11 µm of the flagellum, which is called the midpiece, or alternately, the middle piece. Where the midpiece terminates, a second sheath, called the fibrous sheath (FS), covers the next portion of the flagellum, which is called the principal piece as can be seen in Figure 1B–D. The sheath in the principal piece extends for ∼40 µm in bull sperm. In all mammalian sperm, the FS tapers in thickness becoming progressively thinner in the distal direction. It does not extend to the last few microns of the flagellum. The sheath becomes greatly reduced and the ODFs are absent in the distal portion of the flagellum, a region called the end piece that can be seen in Figure 1E.

The MS and the FS provide additional mechanical support to the flagellum and increase its stiffness. Since the FS and ODFs taper, the flagellum is not uniform in stiffness, becoming progressively less stiff in the distal region. In addition to the mechanical role of the sheath and ODFs, it is also well documented that the sheath and ODFs are also the site of many enzymes that support metabolism and signaling in mammalian sperm (Vijayaraghavan et al., 1997 Eddy, 2007 ), but these functions are beyond the scope of this review. Much of the initial description of the unique ultrastructural anatomy of the mammalian sperm was first compiled by Fawcett and Phillips (Fawcett, 1958, 1975 Fawcett and Phillips, 1969, 1970 Phillips, 1972, 1997 ). An early synthesis of the relationship of anatomy to function was published by Phillips and Olson ( 1973 ), which built a foundation for our current understanding.

Spontaneous Generation

Learning Objectives

Explain the theory of spontaneous generation and why people once accepted it as an explanation for the existence of certain types of organisms

Explain how certain individuals (van Helmont, Redi, Needham, Spallanzani, and Pasteur) tried to prove or disprove spontaneous generation

Humans have been asking for millennia: Where does new life come from? Religion, philosophy, and science have all wrestled with this question. One of the oldest explanations was the theory of spontaneous generation, which can be traced back to the ancient Greeks and was widely accepted through the Middle Ages.

The Theory of Spontaneous Generation

The Greek philosopher Aristotle (384–322 BC) was one of the earliest recorded scholars to articulate the theory of spontaneous generation , the notion that life can arise from nonliving matter. Aristotle proposed that life arose from nonliving material if the material contained pneuma (“vital heat”). As evidence, he noted several instances of the appearance of animals from environments previously devoid of such animals, such as the seemingly sudden appearance of fish in a new puddle of water. [1]

This theory persisted into the 17th century, when scientists undertook additional experimentation to support or disprove it. By this time, the proponents of the theory cited how frogs simply seem to appear along the muddy banks of the Nile River in Egypt during the annual flooding. Others observed that mice simply appeared among grain stored in barns with thatched roofs. When the roof leaked and the grain molded, mice appeared. Jan Baptista van Helmont, a 17th century Flemish scientist, proposed that mice could arise from rags and wheat kernels left in an open container for 3 weeks. In reality, such habitats provided ideal food sources and shelter for mouse populations to flourish.

However, one of van Helmont’s contemporaries, Italian physician Francesco Redi (1626–1697), performed an experiment in 1668 that was one of the first to refute the idea that maggots (the larvae of flies) spontaneously generate on meat left out in the open air. He predicted that preventing flies from having direct contact with the meat would also prevent the appearance of maggots. Redi left meat in each of six containers ( Figure 3.2 ). Two were open to the air, two were covered with gauze, and two were tightly sealed. His hypothesis was supported when maggots developed in the uncovered jars, but no maggots appeared in either the gauze-covered or the tightly sealed jars. He concluded that maggots could only form when flies were allowed to lay eggs in the meat, and that the maggots were the offspring of flies, not the product of spontaneous generation.

Figure 3.2 Francesco Redi’s experimental setup consisted of an open container, a container sealed with a cork top, and a container covered in mesh that let in air but not flies. Maggots only appeared on the meat in the open container. However, maggots were also found on the gauze of the gauze-covered container.

In 1745, John Needham (1713–1781) published a report of his own experiments, in which he briefly boiled broth infused with plant or animal matter, hoping to kill all preexisting microbes. [2] He then sealed the flasks. After a few days, Needham observed that the broth had become cloudy and a single drop contained numerous microscopic creatures. He argued that the new microbes must have arisen spontaneously. In reality, however, he likely did not boil the broth enough to kill all preexisting microbes.

Lazzaro Spallanzani (1729–1799) did not agree with Needham’s conclusions, however, and performed hundreds of carefully executed experiments using heated broth. [3] As in Needham’s experiment, broth in sealed jars and unsealed jars was infused with plant and animal matter. Spallanzani’s results contradicted the findings of Needham: Heated but sealed flasks remained clear, without any signs of spontaneous growth, unless the flasks were subsequently opened to the air. This suggested that microbes were introduced into these flasks from the air. In response to Spallanzani’s findings, Needham argued that life originates from a “life force” that was destroyed during Spallanzani’s extended boiling. Any subsequent sealing of the flasks then prevented new life force from entering and causing spontaneous generation ( Figure 3.3 ).

E. Capanna. “Lazzaro Spallanzani: At the Roots of Modern Biology.” Journal of Experimental Zoology 285 no. 3 (1999):178–196.

R. Mancini, M. Nigro, G. Ippolito. “Lazzaro Spallanzani and His Refutation of the Theory of Spontaneous Generation.” Le Infezioni in Medicina 15 no. 3 (2007):199–206.

Figure 3.3 (a) Francesco Redi, who demonstrated that maggots were the offspring of flies, not products of spontaneous generation. (b) John Needham, who argued that microbes arose spontaneously in broth from a “life force.” (c) Lazzaro Spallanzani, whose experiments with broth aimed to disprove those of Needham.

Describe the theory of spontaneous generation and some of the arguments used to support it.

Explain how the experiments of Redi and Spallanzani challenged the theory of spontaneous generation.

Disproving Spontaneous Generation

The debate over spontaneous generation continued well into the 19th century, with scientists serving as proponents of both sides. To settle the debate, the Paris Academy of Sciences offered a prize for resolution of the problem. Louis Pasteur, a prominent French chemist who had been studying microbial fermentation and the causes of wine spoilage, accepted the challenge. In 1858, Pasteur filtered air through a gun-cotton filter and, upon microscopic examination of the cotton, found it full of microorganisms, suggesting that the exposure of a broth to air was not introducing a “life force” to the broth but rather airborne microorganisms.

Later, Pasteur made a series of flasks with long, twisted necks (“swan-neck” flasks), in which he boiled broth to sterilize it ( Figure 3.4 ). His design allowed air inside the flasks to be exchanged with air from the outside, but prevented the introduction of any airborne microorganisms, which would get caught in the twists and bends of the flasks’ necks. If a life force besides the airborne microorganisms were responsible for microbial growth within the sterilized flasks, it would have access to the broth, whereas the microorganisms would not. He correctly predicted that sterilized broth in his swan-neck flasks would remain sterile as long as the swan necks remained intact. However, should the necks be broken, microorganisms would be introduced, contaminating the flasks and allowing microbial growth within the broth.

Pasteur’s set of experiments irrefutably disproved the theory of spontaneous generation and earned him the prestigious Alhumbert Prize from the Paris Academy of Sciences in 1862. In a subsequent lecture in 1864, Pasteur articulated “ Omne vivum ex vivo ” (“Life only comes from life”). In this lecture, Pasteur recounted his famous swan- neck flask experiment, stating that “…life is a germ and a germ is life. Never will the doctrine of spontaneous generation recover from the mortal blow of this simple experiment.” [4] To Pasteur’s credit, it never has.

R. Vallery-Radot. The Life of Pasteur , trans. R.L. Devonshire. New York: McClure, Phillips and Co, 1902, 1:142.

Figure 3.4 (a) French scientist Louis Pasteur, who definitively refuted the long-disputed theory of spontaneous generation. (b) The unique swan-neck feature of the flasks used in Pasteur’s experiment allowed air to enter the flask but prevented the entry of bacterial and fungal spores. (c) Pasteur’s experiment consisted of two parts. In the first part, the broth in the flask was boiled to sterilize it. When this broth was cooled, it remained free of contamination. In the second part of the experiment, the flask was boiled and then the neck was broken off. The broth in this flask became contaminated. (credit b: modification of work by “Wellcome Images”/Wikimedia Commons)

How did Pasteur’s experimental design allow air, but not microbes, to enter, and why was this important?

What was the control group in Pasteur’s experiment and what did it show?


Receptor methylation plays an integral role in bacterial chemotaxis. With the notable exception of Helicobacter pylori (Pittman et al., 2001), all flagellated bacteria appear to employ some form of receptor methylation for chemotaxis ʊlexander & Zhulin, 2007 Wuichet et al., 2007), although the mechanistic details can vary significantly across species. Despite its prevalence, receptor methylation has only been extensively studied so far in E. coli. Little is known about this mechanism in other species of bacteria. In this work, we explored how covalent modification of the adaptation sites affects chemotaxis signalling in B. subtilis. The canonical asparagine receptor, McpB, has three adaptation sites, located at residues 371, 630 and 637 (Zimmer et al., 2000). We found that the amidation of site 371 increased the sensitivity (i.e. binding affinity) of the receptor to asparagine whereas the corresponding changes to sites 630 and 637 had no substantive effect. We also found that the amidation of the negatively charged glutamates at sites 630 and 637 decreased kinase activity whereas the amidation of the glutamate at site 371, with a single exception, increased kinase activity (Table  2 ). Finally, the electrostatic surface potential of the cytoplasmic domain of McpB showed that negative charges at the adaptation sites might further explain the effect of covalent modifications on chemotactic ability.

The B. subtilis strains used in this study all contain CheD, which functions in the CheC˽/Y adaptation system by interacting with CheC when CheYp levels are high, effectively recruiting CheD away from the receptors. Past experiments have shown that mutants lacking cheD activate CheA kinase poorly (Kirby et al., 2001). It is possible that the glutamate and glutamine substitutions at the three methylation sites may change the receptor's affinity for CheD, thereby influencing CheA kinase activity. Thus, the net kinase activity in the cell could reflect not only the direct change in the receptor caused by covalent modification of the adaptation sites but also the secondary effect of changing the receptor's affinity for CheD. However, preliminary experiments indicate that if there are such changes in affinity, they are slight.

As mentioned in Results, the reason that we were able to use chemotaxis assays to study receptor methylation is that there are two other adaptation systems in B. subtilis, the CheC˽/Y and CheV systems. These systems are redundant, with two sufficient for chemotaxis, albeit at reduced efficiency (Rao et al., 2008). In our experiments, the methylation system was inactivated. Nonetheless, the cells were still able to migrate up gradients of attractant although with varying efficiencies based on the modification state ʏig.  2 ). Moreover, the cells were incapable of perfect adaptation (Table  2 ).

Role of electostatic interactions

As outlined in Results, the presence of negative charges (glutamates) at sites 630 and 637 greatly increases the surface negative potential of the adaptation region ʏig.  3 ). Based on a model proposed by the Falke laboratory for the E. coli receptors, we hypothesized that these electrostatic interactions probably affect the intra- and inter-subunit packing of the B. subtilis receptors. However, the receptor–kinase complexes in B. subtilis and E. coli have reciprocal polarity. Attractant binding increases kinase activity in B. subtilis whereas it inhibits it in E. coli. This reciprocal polarity would potentially suggest that the E. coli model cannot be fully applied to B. subtilis. However, the differences in kinase activation most likely are due to how the two sets of receptors interact with the kinase and not to the receptors themselves. Evidence comes from dynamic receptor localization studies, where the binding of attractant has been shown to disrupt receptor packing in both B. subtilis and E. coli (Lamanna et al., 2005). The packing was restored once the cells adapted to the attractant, presumably due in part to methylation and the concomitant neutralization of repulsive electrostatic interactions. Moreover, these results demonstrate that despite the differences in polarity, the same changes in receptor packing are observed in the two species of bacteria. Also, they demonstrate that these same changes lead alternatively to kinase activation in B. subtilis and kinase inhibition in E. coli, providing further evidence that the differences are due to how the receptors interact with the kinase and not the adaptation region, which directly affects packing. Finally, these patterns of reorganization are also consistent with our model where repulsive interactions between negatively charged glutamates at sites 630 and 637 destabilize the receptor and lead to increased kinase activity.

Dissimilarity between B. subtilis and E. coli is also intrinsic to the receptors themselves. In E. coli, substitutions of glutamines or glutamates at all methylatable positions both increase the kinase activity and the 𠆊pparent Kd’ of the receptor (Li & Weis, 2000 Sourjik & Berg, 2002a). In B. subtilis, however, we found that the modification state of sites 630 and 637 affects only the kinase activity but not the 𠆊pparent Kd’. Only the amidation state of site 371 affects the 𠆊pparent Kd’. Interestingly, we found in our structural modelling that the amidation state of site 371 does not affect the surface potential as greatly as amidation states of sites 630 and 637.

Implication of activation and sensitivity being decoupled from one another

As mentioned earlier, activity and sensitivity are not directly correlated with one another in McpB. In particular, there are modifications with high activity and high apparent affinity 𨍱Q��) and others with low activity and low apparent affinity �𯘰Q� and 371E�𯘷Q). Likewise, there are modifications with high activity and low apparent affinity ���) and perhaps even one with low activity and high apparent affinity 𨍱Q𯘰Q𯘷Q). In E. coli, on the other hand, there is a direct, inverse correlation between the two (Sourjik & Berg, 2002b, 2004). In the models commonly used to explain receptor activity in E. coli, the receptor complex is assumed to exist in one of two states: ʁ) a high-affinity, low-activity state and ʂ) a low-affinity, high-activity state (Keymer et al., 2006 Mello & Tu, 2005 Rao et al., 2004). The equilibrium partitioning between these two states is determined by the concentration of chemoattractant and degree of methylation˺midation. Our data for McpB, however, imply that the mechanism for receptor activation cannot be described by a simple two-state model but instead requires a more complicated model involving additional conformational states. While we still lack the requisite biochemical data to construct such a quantitative model, our results nonetheless suggest that B. subtilis is able to independently tune these two factors.

Receptor structure

How are activity and sensitivity decoupled from one another? In particular, why do modifications to site 371 affect the 𠆊pparent Kd’ whereas ones to site 630 and 637 do not? While the actual mechanism is still unknown, we note that the associated mechanism of attractant binding is different in E. coli and B. subtilis. In E. coli, attractants bind across the dimer interface and induce a piston-like movement in the descending helix, the one emerging from the transmembrane region ʌhervitz & Falke, 1996 Yeh et al., 1993). In B. subtilis, attractant binds within an individual monomer and induces a rotation between the helices (Glekas et al., 2010 Szurmant et al., 2004). Site 371 is located on the descending helix. Thus, modifications to it may affect the ‘information flow’ towards both the sensing domain and the kinase, located at the turn (𠆋ottom’) of the receptor ʏig.  4 ). By contrast, sites 630 and 637 are on the ascending helix. Modifications to these sites may affect just the information flow solely towards the kinase ʏig.  4 ). In the three-dimensional structural model, of course, the three sites appear to form a closely spaced triad. The close proximity of the sites suggests that they may be affected by electrostatic interactions between them. It also suggests that modification of each site may affect not only the conformation of each monomer of the cytoplasmic domain separately but also the interface of the two monomers that form the tightly wound dimer. Finally, comparing a sequence alignment of other B. subtilis receptors such as McpA and McpC, we find that the triad of putative methylation sites, and presumably mode of action, is conserved in other B. subtilis receptors (Le Moual & Koshland, 1996).

Schematic representation of the McpB chemoreceptor monomer. The three adaptation sites are shown as white boxes. The cartoon shows that site 371 is in close proximity to the HAMP ( h istidine kinase, a denyl cyclase, m ethyl-accepting chemotaxis protein and p hosphatase) domain ʊravind & Ponting, 1999), which can transmit signals to ʊnd from) the sensing domain, the site where the ligand binds. Sites 630 and 637 can affect the signalling domain, which interacts with the kinase.

Comparison with previous work

Previously it was argued that the modification, amidation or methylation, of site 630 increased kinase activity whereas the modification of site 637 decreased it. These results were obtained from experiments where aspartate substitutions at each of the three sites and in combinations were examined using the tethered cell assay (Zimmer et al., 2000). This approach was based on previous work from the Koshland lab (Shapiro & Koshland, 1994), in which glutamate/glutamine residues were substituted with aspartate residues where a ‘permanent’ negative charge would be at the sites that could not be neutralized by methylation. Based on these previous studies, we anticipated that there would be some difference between sites 630 and 637 in the experiments reported in Tables  1 or ​ or2, 2 , but evidently there is none. One possible explanation for this discrepancy is that previously employed aspartate-for-glutamate/glutamine substitutions, which are shorter by one methylene group, may alter the conformation of the receptor in some unnatural way. Considering the close proximity of the three adaptation sites, it does seem plausible that moving the negative charge from its position in a glutamate to its position in an aspartate (the distance of a methylene group or 1.33 Å) could have unnatural effects.

Model for site-specific methylation during taxis in a concentration gradient of attractant

Based on the results of this work, we propose the following model for site-specific methylation. In the absence of attractant, we expect that site 371 is either amidated (i.e. glutamine) or methylated and sites 630 and 637 are unmethylated (i.e. glutamates). Such a modification state would be optimal in the sense that the kinase is maximally active and the 𠆊pparent Kd’ lowest. Thus, the bacteria would be able to detect a concentration gradient of attractant beginning at quite low concentrations. As the bacterium swam up the gradient, site 371 would gradually be deamidated when it is a glutamine or demethylated when a methyl-glutamate. This would have the effect of reducing kinase activity due to higher ambient concentrations of asparagine as part of the adaptation process and also increasing the 𠆊pparent Kd’, enabling the bacterium to optimally sense gradients at higher concentrations of attractant. Similarly, we expect that sites 630 and 637 would gradually become more methylated ʏor which amidation was used in this study as a mimic). This would have the effect of further reducing kinase activity as part of the adaptation process. Based on the relative timing of the demethylation and methylation steps (Kirby et al., 1999), we expect that changes at site 371 would occur more rapidly than those at sites 630 and 637. The reason why these two processes occur on different timescales, however, is still unknown.


In summary, we have found that amidation of site 371 increases McpB's apparent affinity for asparagine and also, in most cases, increases kinase activity. In addition, we found that amidation of sites 630 and 637 decreases kinase activity but does not affect the apparent affinity. These findings further our understanding of the site-specific methylation system in B. subtilis by demonstrating how the modification of specific sites can have varying effects on receptor function.


Prokaryotic cells move through liquids or over moist surfaces by swimming, swarming, gliding, twitching or floating. An impressive diversity of motility mechanisms has evolved in prokaryotes. Movement can involve surface appendages, such as flagella that spin, pili that pull and Mycoplasma 'legs' that walk. Internal structures, such as the cytoskeleton and gas vesicles, are involved in some types of motility, whereas the mechanisms of some other types of movement remain mysterious. Regardless of the type of motility machinery that is employed, most motile microorganisms use complex sensory systems to control their movements in response to stimuli, which allows them to migrate to optimal environments.


Multisubunit protein complexes are ubiquitous in biology and perform a plethora of essential functions. Most of the scientific literature treats such assemblies as static: their function is assumed to be independent of their manner of assembly, and their structure is assumed to remain intact until they are degraded. Recent observations of the bacterial flagellar motor, among others, bring these notions into question. The torque-generating stator units of the motor assemble and disassemble in response to changes in load. Here, we used electrorotation to drive tethered cells forward, which decreases motor load, and measured the resulting stator dynamics. No disassembly occurred while the torque remained high, but all of the stator units were released when the motor was spun near the zero-torque speed. When the electrorotation was turned off, so that the load was again high, stator units were recruited, increasing motor speed in a stepwise fashion. A model in which speed affects the binding rate and torque affects the free energy of bound stator units captures the observed torque-dependent stator assembly dynamics, providing a quantitative framework for the environmentally regulated self-assembly of a major macromolecular machine.

Biology is replete with examples of macromolecular protein complexes, which consist of smaller components that self-assemble to form functional molecular machines (1). Such machines perform essential biological functions across life forms, such as protein synthesis, ATP production, DNA replication, and intracellular transport (2 ⇓ ⇓ –5). The assembly of such complexes is known to be regulated at the level of gene transcription and protein synthesis, but little is known about the factors that control the fate of the assembly once the mature protein subunits enter their target space (cytoplasm, membrane, or cell wall). Typically, assembled protein complexes are assumed to be static, with functions independent of their mode of assembly.

A growing body of literature on subunit exchange in protein complexes is bringing this worldview into question (6). Among these, the bacterial flagellar motor, e.g., of Escherichia coli (Fig. 1A), has emerged as a prime example of a macromolecular complex whose assembly is dynamically modulated in a functionally relevant manner and serves as a case study in which a quantitative description of the process can be rigorously laid out. Self-assembled at the cell wall from over 20 different kinds of proteins, this motor propels cells through fluids by rotating extracellular helical filaments (7, 8). The part of the motor embedded in the inner membrane is called the rotor. Torque-generating stator units (each consisting of four MotA and two MotB proteins) bind to the peptidoglycan layer and apply torque on the rotor (9, 10). Up to 11 stator units work together to drive the motor and the bound units exchange with an inner membrane-embedded pool of unbound units (11 ⇓ –13). The motor adapts to changes in the mechanical load by changing the number of stator units, thereby matching output with demand (14 ⇓ –16). This dynamic self-assembly enables the cell to conserve resources. For example, when motors are first assembled and flagellar filaments are short, the torque required to spin them can be supplied by a small number of stator units, each of which passes the same number of protons per revolution (assuming tight coupling). A larger number of units waste energy without improving function.

(A) Schematic of the flagellar motor of E. coli. Helical filaments that propel the cell are driven at their base by the motor. A flexible hook connects the filament to the motor’s drive shaft, which passes through the L ring (in the outer or lipopolysaccharide membrane) and the P ring (at the peptidoglycan layer) to reach the rotor (green, in the inner or cytoplasmic membrane). Stator units (red) bind to the peptidoglycan layer, span the cytoplasmic membrane, and apply torque on the C ring (at the level of the horizontal dashed line) to drive the motor. Several stator units work together to drive the motor at any time, as shown in the cross-sectional view. (B) The cell is tethered to a surface via a short flagellar stub. The motor rotates the cell body and exerts a high torque, as depicted by the lower left black arrow. We apply an assistive electrorotation torque (green) on the cell via a high-frequency rotating electric field it spins the cell at high speed and reduces the motor torque (lower right). (C) A torque–speed curve for a motor with 10 stator units. The progress of the experiment, from points 1, 2, 3, 4 and back to 1, is described in the text. The motor loses 4 stator units between 2 and 3 and recruits an equal number between 4 and 1. The torque–speed curves for motors with fewer than 10 stator units are shown by the dotted lines. In moving from 1 to 2, the torque drops gradually from 1 to the knee and then rapidly from the knee to 2. If the electrorotation field is strong enough, the rotation reduces the torque to zero (at the zero-torque speed).

Here we report the precise dependence of stator stoichiometry on torque over the full range of operating conditions, at steady state as well as after sudden changes in motor torque. To control the torque, we used electrorotation (Fig. 1B), in which a fast-rotating electric field applies external torque on a tethered cell (17, 18). Cells were tethered to sapphire via a short sticky-filament stub, and the rotating electric field was applied using an apparatus developed earlier (Materials and Methods). When the external field was turned on, the cell rapidly sped up. We measured the dynamics of stator remodeling following a change in motor rotation rate from low speeds of about 10 Hz to high speeds ranging from 50 Hz to 300 Hz. The torque produced by the motor over these speeds ranged from high torque at low speeds to zero torque (and occasionally negative torque) at 300 Hz. The motor released all its stator units at speeds near the zero-torque speed. When the external field was turned off, the external load returned to a large value, and the speed increased in a stepwise manner, as new stator units were recruited. We used these measurements and the tools of statistical physics to develop a model for the torque-dependent stator assembly, which captured the observed dynamics.

Why do flagella form a bundle only when they rotate counterclockwise during chemotaxis? - Biology

The bacterial flagellar motor is a molecular machine that converts an ion flux to the rotation of a helical flagellar filament. Counterclockwise rotation of the filaments allows them to join in a bundle and propel the cell forward. Loss of motility can be caused by environmental factors such as temperature, pH, and solvation. Hydrostatic pressure is also a physical inhibitor of bacterial motility, but the detailed mechanism of this inhibition is still unknown. Here, we developed a high-pressure microscope that enables us to acquire high-resolution microscopic images, regardless of applied pressures. We also characterized the pressure dependence of the motility of swimming Escherichia coli cells and the rotation of single flagellar motors. The fraction and speed of swimming cells decreased with increased pressure. At 80 MPa, all cells stopped swimming and simply diffused in solution. After the release of pressure, most cells immediately recovered their initial motility. Direct observation of the motility of single flagellar motors revealed that at 80 MPa, the motors generate torque that should be sufficient to join rotating filaments in a bundle. The discrepancy in the behavior of free swimming cells and individual motors could be due to the applied pressure inhibiting the formation of rotating filament bundles that can propel the cell body in an aqueous environment.

Masayoshi Nishiyama's present address is The Hakubi Center, Kyoto University, Kyoto, Japan.

Cooperation and Communication

Bacteria cooperate when cells perform actions that benefit other cells or the entire colony, and these actions are selected for [8]. Bacteria have developed cooperative behavior to cope with difficult environmental conditions. Bacteria communicate between individual cells and with the entire colony in order to cooperatively form patterns [2]. Generally, a higher lever of cooperation is observed when conditions are less favorable, such as in high-agar or low-nutrient media [4].

Bacteria colonies can be through of as multicellular organisms for the purposes of identifying patterns of growth [1]. Bacteria are able to change the morphotype of their entire colony (for example, from branching to chiral) within a time period as short as 48 hours in order to better suit their environment [2]. The ability of the colony to adhere to one morphotype and to transition completely to another are both characteristics of cooperative multicellular behavior and intercellular communication.

Bacteria communicate with other cells within the colony using a variety of methods, including direct and indirect cell-cell physical and chemical interactions, long range chemical signaling, and chemotactic signaling. The production of wetting fluid is an example of an indirect physical interaction [2]. Examples of chemical interactions include: long-range and short-range chemorepulsion (movement of a cell away from a substance), short-range chemoattraction (movement of a cell towards a substance), and rotational chemotaxis (movement of a cell guided by a chemical concentration gradient) [7].

Watch the video: Chemotaxis Assays Using the ibidi µ-Slide Chemotaxis (December 2021).