Reversal of Flagellar Rotation Is Important in Initial Attachment of Escherichia coli to Glass in a Dynamic System with High- and Low-Ionic-Strength Buffers

The attachment rates of wild-type, smooth-swimming, tumbly, and paralyzed Escherichia coli to glass was measured at fluid velocities of 0.0044 and 0.044 cms⁻¹ (corresponding to shear rates of 0.34 and 3.4 s⁻¹, respectively), in 0.02 and 0.2 M buffer solutions. At the highest ionic strength, we did not observe a significant difference in the attachment rate of wild-type and paralyzed cells at either fluid velocity. However, when the ionic strength was reduced, paralyzed bacteria attached at rates 4 and 10 times lower than that of the wild type under fluid velocities of 0.0044 and 0.044 cms⁻¹, respectively. This suggested that the rotation of the flagella assisted in attachment. We then compared the attachment rates of smooth-swimming (counterclockwise rotation only) and tumbly (clockwise rotation only) cells to the wild type to determine whether the direction of rotation was important to cell attachment. At 0.0044 cms⁻¹, the smooth-swimming cells attached at rates similar to that of the wild type in both buffer solutions but significantly less at the higher fluid velocity. Tumbly cells attached at much lower rates under all conditions. Thus, the combination of clockwise and counterclockwise flagellar rotation and their coupling appeared to be important in cell attachment. We considered a number of hypotheses to interpret these observations, including a residence time analysis and a comparison of traditional Derjaguin-Landau-Verwey-Overbeek (DLVO) theory to soft-particle theory.     

flaAII (motC, cheV) of Salmonella typhimurium is a structural gene involved in energization and switching of the flagellar motor.

The flaAII gene of Salmonella typhimurium has also been termed motC and cheV, because defective alleles may give rise to a nonflagellate, paralyzed, or nonchemotactic phenotype. We isolated a temperature-sensitive motility mutant (MY1) and have found that the mutation occurs in the flaAII gene. In temperature-jump experiments, MY1 could be converted from highly motile to paralyzed within 0.5 s, demonstrating that flaAII is a structural gene whose product is immediately essential for motor rotation. The mutant, although chemotactic at permissive temperatures (less than 36 degrees C), had a higher clockwise rotational bias than did the wild type; it can therefore be regarded simultaneously as motC(Ts) and cheV (tumbly). The only previously reported S. typhimurium cheV mutant was smooth-swimming. A shift toward counterclockwise bias accompanied loss of rotational speed in the restrictive temperature range. This result, by analogy with known proton motive force effects on motor switching, further indicates a central role of the flaAII (motC, cheV) protein in the energy transduction and switching process. Since there is no evidence associating it with the isolable entity known as the basal body, it may reside at the cytoplasmic face of the flagellar motor.     

Coordination of flagella on filamentous cells of Escherichia coli.

Video techniques were used to study the coordination of different flagella on single filamentous cells of Escherichia coli. Filamentous, nonseptate cells were produced by introducing a cell division mutation into a strain that was polyhook but otherwise wild type for chemotaxis. Markers for its flagellar motors (ordinary polyhook cells that had been fixed with glutaraldehyde) were attached with antihook antibodies. The markers were driven alternately clockwise and counterclockwise, at angular velocities comparable to those observed when wild-type cells are tethered to glass. The directions of rotation of different markers on the same cell were not correlated; reversals of the flagellar motors occurred asynchronously. The bias of the motors (the fraction of time spent spinning counterclockwise) changed with time. Variations in bias were correlated, provided that the motors were within a few micrometers of one another. Thus, although the directions of rotation of flagellar motors are not controlled by a common intracellular signal, their biases are. This signal appears to have a limited range.     

Flagella and motility behaviour of square bacteria.

Square bacteria are shown to have right-handed helical (RH) flagella. They swim forward by clockwise (CW), and backwards by counterclockwise (CCW) rotation of their flagella. They are propelled by several or single filaments arising at several or single points on the cell surface. When there are several filaments a stable bundle is formed that does not fly apart during the change from clockwise to counterclockwise rotation or vice versa. In addition to the flagella attached to the cells, large amounts of detached flagella aggregated into thick super-flagella, can be observed at all phases of growth.     

Subdivision of flagellar genes of Salmonella typhimurium into regions responsible for assembly, rotation, and switching.

Three flagellar genes of Salmonella typhimurium (flaAII.2, flaQ, and flaN) were found to be multifunctional, each being associated with four distinct mutant phenotypes: nonflagellate (Fla-), paralyzed (Mot-), nonchemotactic (Che-) with clockwise motor bias, and nonchemotactic (Che-) with counterclockwise motor bias. The distribution of Fla, Mot, and Che mutational sites within each gene was examined. Fla sites were fairly broadly distributed, whereas Mot and Che sites were more narrowly defined. Local subregions rich in sites of one type were not generally rich in sites of another type. Among Che sites, there was little overlap between those corresponding to a clockwise bias and those corresponding to a counterclockwise bias. Our results suggest that within the corresponding gene products there are specialized subregions for flagellar structure, motor rotation, and control of the sense of rotation.     

Microcinematographic analysis of tethered Leptospira illini.

A model of Leptospira motility was recently proposed. One element of the model states that in translating cells the anterior spiral-shaped end gyrates counterclockwise and the posterior hook-shaped end gyrates clockwise. We tested these predictions by analyzing cells tethered to a glass surface. Leptospira illini was incubated with antibody-coated latex beads (Ab-beads). These beads adhered to the cells, and subsequently some cells became attached to either the slide or the cover glass via the Ab-beads. As previously reported, these cells rapidly moved back and forth across the surface of the beads. In addition, a general trend was observed: cells tethered to the cover glass rotated clockwise around the Ab-bead; cells tethered to the slide rotated counterclockwise around the Ab-bead. A computer-aided microcinematographic analysis of tethered cells indicated that the direction of rotation of cells around the Ab-bead was a function of both the surface of attachment and the shape of the cell ends. The results can best be explained by assuming that the gyrating ends interact with the glass surface to cause rotation around the Ab-beads. The analysis obtained indicates that the hook- and spiral-shaped ends rotate in the directions predicted by the model. In addition, the tethered cell assay permitted detection of rapid, coordinated reversals of the cell ends, e.g., cells rapidly switched from a hook-spiral configuration to a spiral-hook configuration. These results suggest the existance of a mechanism which coordinates the shape of the cell ends of L. illini.     

Cell envelopes of chemotaxis mutants of Escherichia coli rotate their flagella counterclockwise.

Flagella rotated exclusively counterclockwise in Escherichia coli cell envelopes prepared from wild-type cells, whose flagella rotated both clockwise and counterclockwise, from mutants rotating their flagella counterclockwise only, and even from mutants rotating their flagella primarily clockwise. Some factor needed for clockwise flagellar rotation appeared to be missing or defective in the cell envelopes.     

Switching of the Bacterial Flagellar Motor Near Zero Load

Flagellated bacteria, such as Escherichia coli, are able to swim up gradients of chemical attractants by modulating the direction of rotation of their flagellar motors, which spin alternately clockwise (CW) and counterclockwise (CCW). Chemotactic behavior has been studied under a variety of conditions, mostly at high loads (at large motor torques). Here, we examine motor switching at low loads. Nano-gold spheres of various sizes were attached to hooks (the flexible coupling at the base of the flagellar filament) of cells lacking flagellar filaments in media containing different concentrations of the viscous agent Ficoll. The speeds and directions of rotation of the spheres were measured. Contrary to the case at high loads, motor switching rates increased appreciably with load. Both the CW → CCW and CCW → CW switching rates increased linearly with motor torque. Evidently, the switch senses stator-rotor interactions as well as the CheY-P concentration.     

Pausing of flagellar rotation is a component of bacterial motility and chemotaxis.

When bacterial cells are tethered to glass by their flagella, many of them spin. On the basis of experiments with tethered cells it has generally been thought that the motor which drives the flagellum is a two-state device, existing in either a counterclockwise or a clockwise state. Here we show that a third state of the motor is that of pausing, the duration and frequency of which are affected by chemotactic stimuli. We have recorded on video tape the rotation of tethered Escherichia coli and Salmonella typhimurium cells and analyzed the recordings frame by frame and in slow motion. Most wild-type cells paused intermittently. The addition of repellents caused an increase in the frequency and duration of the pauses. The addition of attractants sharply reduced the number of pauses. A chemotaxis mutant which lacks a large part of the chemotaxis machinery owing to a deletion of the genes from cheA to cheZ did not pause at all and did not respond to repellents by pausing. A tumbly mutant of S. typhimurium responded to repellents by smooth swimming and to attractants by tumbling. When tethered, these cells exhibited a normal rotational response but an inverse pausing response to chemotactic stimuli: the frequency of pauses decreased in response to repellents and increased in response to attractants. It is suggested that (i) pausing is an integral part of bacterial motility and chemotaxis, (ii) pausing is independent of the direction of flagellar rotation, and (iii) pausing may be one of the causes of tumbling.     

Spiral growth in the radially-expanding piloboloid mutants ofPhycomyces blakesleeanus

The growth zone of the sporangiophore of a piloboloid mutant,pil, ofPhycomyces expands radially at an increased rate until the growth zone becomes nearly spherical, in sharp contrast to that of the wild-type sporangiophore which exhibits longitudinal elongation only and is conical. The rotation of thepil sporangiophore reverses its direction from clockwise (CW) to counterclockwise (CCW) during the period of increased radial expansion, and the CCW rotation continues as long as does the radial expansion. The direction of rotation and the time of reversal are correlated with the relative rates of cell-wall expansion in the longitudinal and transverse directions. The CCW rotation of the sporangiophore of this mutant can be explained by the behavior of the microfibrils, as previously proposed to explain the rotation of the wild-type sporangiophore.Abbreviations CW clockwise - CCW counterclockwise — both as viewed from above     

Regulation of Switching Frequency and Bias of the Bacterial Flagellar Motor by CheY and Fumarate

The effect of CheY and fumarate on switching frequency and rotational bias of the bacterial flagellar motor was analyzed by computer-aided tracking of tethered Escherichia coli. Plots of cells overexpressing CheY in a gutted background showed a bell-shaped correlation curve of switching frequency and bias centering at about 50% clockwise rotation. Gutted cells (i.e., with cheA to cheZ deleted) with a low CheY level but a high cytoplasmic fumarate concentration displayed the same correlation of switching frequency and bias as cells overexpressing CheY at the wild-type fumarate level. Hence, a high fumarate level can phenotypically mimic CheY overexpression by simultaneously changing the switching frequency and the bias. A linear correlation of cytoplasmic fumarate concentration and clockwise rotation bias was found and predicts exclusively counterclockwise rotation without switching when fumarate is absent. This suggests that (i) fumarate is essential for clockwise rotation in vivo and (ii) any metabolically induced fluctuation of its cytoplasmic concentration will result in a transient change in bias and switching probability. A high fumarate level resulted in a dose-response curve linking bias and cytoplasmic CheY concentration that was offset but with a slope similar to that for a low fumarate level. It is concluded that fumarate and CheY act additively presumably at different reaction steps in the conformational transition of the switch complex from counterclockwise to clockwise motor rotation.     

Asynchronous switching of flagellar motors on a single bacterial cell

Salmonella possesses several flagella, each capable of counterclockwise and clockwise rotation. Counterclockwise rotation produces swimming, clockwise rotation produces tumbling. Switching between senses occurs stochastically. The rotational sense of individual flagella on a single cell could be monitored under special conditions (partially de-energized cells of cheC and cheZ mutants). Switching was totally asynchronous, indicating that the stochastic process operates at the level of the individual organelle. Coordinated rotation in the flagellar bundle during swimming may therefore derive simply from a high counterclockwise probability enhanced by mechanical interactions, and not from a synchronizing switch mechanism. Different flagella on a given cell had different switching probabilities, on a time scale (greater than 2 min) spanning many switching events. This heterogeneity may reflect permanent structural differences, or slow fluctuations in some regulatory process.     

Fumarate or a fumarate metabolite restores switching ability to rotating flagella of bacterial envelopes.

Flagella of cytoplasm-free envelopes of Escherichia coli or Salmonella typhimurium can rotate in either the counterclockwise or clockwise direction, but they never switch from one direction of rotation to another. Exogenous fumarate, in the intracellular presence of the chemotaxis protein CheY, restored switching ability to envelopes, with a concomitant increase in clockwise rotation. An increase in clockwise rotation was also observed after fumarate was added to partially lysed cells of E. coli, but the proportion of switching cells remained unchanged.     

Specific inactivator of flagellar reversal in Salmonella typhimurium.

Specific inhibition of flagellar rotation reversal was observed after exposure of chemotactic Salmonella typhimurium to citrate autoclaved at neutral pH. The presence of a rotation reversal inactivator was established in autoclaved citrate-containing media and nutrient broth. Since modulation of flagellar rotation by attractants and repellents is the basis of chemotactic behavior, a specific inhibitor of rotation reversal, which is essential for tumble generation, provides a useful probe into the molecular mechanism of bacterial chemotaxis. The inactivator inhibits clockwise rotation without affecting counterclockwise rotation, speed of rotation, or the capacity of the cells to grow and divide. Inactivation of clockwise rotation is gradual and irreversible, differing from the transient inhibition of clockwise rotation by attractants, which is characterized by an immediate suppression followed by a return to normal rotation patterns. The rotation reversal inactivator is stable to acidification, rotary evaporation, lyophilization, and rehydration.     

Characterizing the adhesion of motile and nonmotile Escherichia coli to a glass surface using a parallel-plate flow chamber

A parallel-plate flow chamber was used to measure the attachment and detachment rates of Escherichia coli to a glass surface at various fluid velocities. The effect of flagella on adhesion was investigated by performing experiments with several E. coli strains: AW405 (motile); HCB136 (nonmotile mutant with paralyzed flagella); and HCB137 (nonmotile mutant without flagella). We compared the total attachment rates and the fraction of bacteria retained on the surface to determine how the presence and movement of the flagella influence transport to the surface and adhesion strength in this dynamic system. At the lower fluid velocities, there was no significant difference in the total attachment rates for the three bacterial strains; nonmotile strains settled at a rate that was of the same order of magnitude as the diffusion rate of the motile strain. At the highest fluid velocity, the effect of settling was minimized to better illustrate the importance of motility, and the attachment rates of both nonmotile strains were approximately five times slower than that of the motile bacteria. Thus, different processes controlled the attachment rate depending on the parameter regime in which the experiment was performed. The fractions of motile bacteria retained on the glass surface increased with increasing velocity, whereas the opposite trend was found for the nonmotile strains. This suggests that the rotation of the flagella enables cells to detach from the surface (at the lower fluid velocities) and strengthens adhesion (at higher fluid velocities), whereas nonmotile cells detach as a result of shear. There was no significant difference in the initial attachment rates of the two nonmotile species, which suggests that merely the presence of flagella was not important in this stage of biofilm development.     

Release of flagellar filament-hook-rod complex by a Salmonella typhimurium mutant defective in the M ring of the basal body.

A Salmonella typhimurium strain possessing a mutation in the fliF gene (coding for the component protein of the M ring of the flagellar basal body) swarmed poorly on a semisolid plate. However, cells grown in liquid medium swam normally and did not show any differences from wild-type cells in terms of swimming speed or tumbling frequency. When mutant cells were grown in a viscous medium, detached bundles of flagellar filaments as long as 100 microns were formed and the cells had impaired motility. Electron microscopy and immunoelectron microscopy revealed that the filaments released from the cells had the hook and a part of the rod of the flagellar basal body still attached. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis showed that the rod portion of the released structures consisted of the 30-kilodalton FlgG protein. Double mutants containing this fliF mutation and various che mutations were constructed, and their behavior in viscous media was analyzed. When the flagellar rotation of the mutants was strongly biased to either a counterclockwise or a clockwise direction, detached bundles were not formed. The formation of large bundles was most extreme in mutants weakly biased to clockwise rotation.     

The Shift-Work Accident Rate is More Related to the Shift Type than to Shift Rotation

The current study investigated the accident rates across morning, late, and night shifts in rotating shift-workers employed in two different shift rotations at the same steel work factory. A retrospective analysis has been performed of accident data (N = 578) over a 5-year period (2003 through 2007) of 730 male shift-workers employed in either a clockwise (mean age of the workers 38.1 ± SD 9.8 years) or counterclockwise rotation (mean age 38.0 ± SD 10.1 years) with comparable work conditions. The overall accident rate across the 24-h day was not significantly different between clockwise and counterclockwise shift rotation. In both shift-work rotations, morning shifts as opposed to night shifts exhibited a significantly higher accident rate. There was no significant difference between late shifts and morning or night shifts in either shift rotation. The increased accident rate in the morning shift at this steel factory could be related to the early starting time of the shift and to this shift being more labor intensive in both shift rotations. These findings suggest that work-related factors must be considered in addition to shift-work schedules when investigating accident rates in rotating shift-workers.     

Both CheA and CheW are required for reconstitution of chemotactic signaling in Escherichia coli.

If cells of Escherichia coli deleted for genes that specify transducers and all known cytoplasmic chemotaxis proteins are reconstituted with CheA, CheW, and CheY, they spin their flagella alternately clockwise and counterclockwise. If the aspartate receptor also is present, clockwise rotation is suppressed upon addition of aspartate. If either CheA or CheW is absent, the fraction of time that the flagella spin clockwise is reduced and responses to aspartate do not occur.     

Reconstitution of signaling in bacterial chemotaxis.

Strains missing several genes required for chemotaxis toward amino acids, peptides, and certain sugars were tethered and their rotational behavior was analyzed. Null strains (called gutted) were deleted for genes that code for the transducers Tsr, Tar, Tap, and Trg and for the cytoplasmic proteins CheA, CheW, CheR, CheB, CheY, and CheZ. Motor switch components were wild type, flaAII(cheC), or flaBII(cheV). Gutted cells with wild-type motors spun exclusively counterclockwise, while those with mutant motors changed their directions of rotation. CheY reduced the bias (the fraction of time that cells spun counterclockwise) in either case. CheZ offset the effect of CheY to an extent that varied with switch allele but did not change the bias when tested alone. Transducers also increased the bias in the presence of CheY but not when tested alone. However, cells containing transducers and CheY failed to respond to attractants or repellents normally detected in the periplasm. This sensitivity was restored by addition of CheA and CheW. Thus, CheY both enhances clockwise rotation and couples the transducers to the flagella. CheZ acts, at the level of the motor, as a CheY antagonist. CheA or CheW or both are required to complete the signal pathway. A model is presented that explains these results and is consistent with other data found in the literature.     

In vitro Approach to Bacterial Chemotaxis

An in vitro approach to study bacterial motility and chemotaxis is described. The approach is based on a preparation of flagellated cell envelopes. The envelopes are prepared from bacteria by a penicillin treatment and subsequent osmotic lysis. When the envelopes are energized, their flagella rotate. The direction of rotation in wild type envelopes is counterclockwise. Inclusion of the CheY protein within the envelopes may restore clockwise rotation. The advantages and disadvantages of this approach are pointed out.