The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus.

Type IV pilus (T4P) systems are complex molecular machines that polymerize major pilin proteins into thin filaments displayed on bacterial surfaces. Pilus functions require rapid extension and depolymerization of the pilus, powered by the assembly and retraction ATPases, respectively. A set of low abundance minor pilins influences pilus dynamics by unknown mechanisms. The Vibrio cholerae toxin-coregulated pilus (TCP) is among the simplest of the T4P systems, having a single minor pilin TcpB and lacking a retraction ATPase. Here we show that TcpB, like its homolog CofB, initiates pilus assembly. TcpB co-localizes with the pili but at extremely low levels, equivalent to one subunit per pilus. We used a micropillars assay to demonstrate that TCP are retractile despite the absence of a retraction ATPase, and that retraction relies on TcpB, as a V. cholerae tcpB Glu5Val mutant is fully piliated but does not induce micropillars movements. This mutant is impaired in TCP-mediated autoagglutination and TcpF secretion, consistent with retraction being required for these functions. We propose that TcpB initiates pilus retraction by incorporating into the growing pilus in a Glu5-dependent manner, which stalls assembly and triggers processive disassembly. These results provide a framework for understanding filament dynamics in more complex T4P systems and the closely related Type II secretion system.

Intact Telopeptides Enhance Interactions between Collagens.

Collagen is the fundamental structural component of a wide range of connective tissues and of the extracellular matrix. It undergoes self-assembly from individual triple-helical proteins into well-ordered fibrils, a process that is key to tissue development and homeostasis, and to processes such as wound healing. Nucleation of this assembly is known to be slowed considerably by pepsin removal of short nonhelical regions that flank collagen's triple helix, known as telopeptides. Using optical tweezers to perform microrheology measurements, we explored the changes in viscoelasticity of solutions of collagen with and without intact telopeptides. Our experiments reveal that intact telopeptides contribute a significant frequency-dependent enhancement of the complex shear modulus. An analytical model of polymers associating to establish chemical equilibrium among higher-order species shows trends in G' and G″ consistent with our experimental observations, including a concentration-dependent crossover in G″/c around 300 Hz. This work suggests that telopeptides facilitate transient intermolecular interactions between collagen proteins, even in the acidic conditions used here.

A Non-Poissonian Flagellar Motor Switch Increases Bacterial Chemotactic Potential.

We investigate bacterial chemotactic strategies using run-tumble and run-reverse-flick motility patterns. The former is typically observed in enteric bacteria such as Escherichia coli and Salmonella and the latter was recently observed in the marine bacteria Vibrio alginolyticus and is possibly exhibited by other polar flagellated species. It is shown that although the three-step motility pattern helps the bacterium to localize near hot spots, an exploitative behavior, its exploratory potential in short times can be significantly enhanced by employing a non-Poissonian regulation scheme for its flagellar motor switches.

From the Cover: Bacterial flagellum as a propeller and as a rudder for efficient chemotaxis.

We investigate swimming and chemotactic behaviors of the polarly flagellated marine bacteria Vibrio alginolyticus in an aqueous medium. Our observations show that V. alginolyticus execute a cyclic, three-step (forward, reverse, and flick) swimming pattern that is distinctively different from the run-tumble pattern adopted by Escherichia coli. Specifically, the bacterium backtracks its forward swimming path when the motor reverses. However, upon resuming forward swimming, the flagellum flicks and a new swimming direction is selected at random. In a chemically homogeneous medium (no attractant or repellent), the consecutive forward t(f) and backward t(b) swimming times are uncorrelated. Interestingly, although t(f) and t(b) are not distributed in a Poissonian fashion, their difference Δt = |t(f) - t(b)| is. Near a point source of attractant, on the other hand, t(f) and t(b) are found to be strongly correlated, and Δt obeys a bimodal distribution. These observations indicate that V. alginolyticus exploit the time-reversal symmetry of forward and backward swimming by using the time difference to regulate their chemotactic behavior. By adopting the three-step cycle, cells of V. alginolyticus are able to quickly respond to a chemical gradient as well as to localize near a point source of attractant.

Implications of three-step swimming patterns in bacterial chemotaxis.

We recently found that marine bacteria Vibrio alginolyticus execute a cyclic three-step (run-reverse-flick) motility pattern that is distinctively different from the two-step (run-tumble) pattern of Escherichia coli. How this novel, to our knowledge, swimming pattern is regulated by cells of V. alginolyticus is not currently known, but its significance for bacterial chemotaxis is self-evident and will be delineated herein. Using a statistical approach, we calculated the migration speed of a cell executing the three-step pattern in a linear chemical gradient, and found that a biphasic chemotactic response arises naturally. The implication of such a response for the cells to adapt to ocean environments and its possible connection to E. coli's response are also discussed.

Purification of Type IV Pili and Pilin Subunits.

The Type IV pili are displayed peritrichously on the surfaces of Neisseria gonorrhoeae cells. Here we present protocols for isolating and purifying Type IV pili and dissociating them into PilE pilin subunits. Pilus filaments are isolated from the bacterial cell surface by mechanical shearing and purified by differential precipitation and centrifugation. PilE subunits are extracted by treating the purified pili with detergent to disrupt the hydrophobic interactions holding them together in the filaments. Purified pili and pilin subunits can be used for structural, biophysical, or biochemical characterization and as antigens for antibody production.

Cryoelectron Microscopy Reconstructions of the Pseudomonas aeruginosa and Neisseria gonorrhoeae Type IV Pili at Sub-nanometer Resolution.

We report here cryoelectron microscopy reconstructions of type IV pili (T4P) from two important human pathogens, Pseudomonas aeruginosa and Neisseria gonorrhoeae, at ∼ 8 and 5 Å resolution, respectively. The two structures reveal distinct arrangements of the pilin globular domains on the pilus surfaces, which impart different helical parameters, but similar packing of the conserved N-terminal α helices, α1, in the filament core. In contrast to the continuous α helix seen in the X-ray crystal structures of the P. aeruginosa and N. gonorrhoeae pilin subunits, α1 in the pilus filaments has a melted segment located between conserved helix-breaking residues Gly14 and Pro22, as seen for the Neisseria meningitidis T4P. Using mutagenesis we show that Pro22 is critical for pilus assembly, as are Thr2 and Glu5, which are positioned to interact in the hydrophobic filament core. These structures provide a framework for understanding T4P assembly, function, and biophysical properties.

An Element of Determinism in a Stochastic Flagellar Motor Switch.

Marine bacterium Vibrio alginolyticus uses a single polar flagellum to navigate in an aqueous environment. Similar to Escherichia coli cells, the polar flagellar motor has two states; when the motor is counter-clockwise, the cell swims forward and when the motor is clockwise, the cell swims backward. V. alginolyticus also incorporates a direction randomization step at the start of the forward swimming interval by flicking its flagellum. To gain an understanding on how the polar flagellar motor switch is regulated, distributions of the forward Δf and backward Δb intervals are investigated herein. We found that the steady-state probability density functions, P(Δf) and P(Δb), of freely swimming bacteria are strongly peaked at a finite time, suggesting that the motor switch is not Poissonian. The short-time inhibition is sufficiently strong and long lasting, i.e., several hundred milliseconds for both intervals, which is readily observed and characterized. Treating motor reversal dynamics as a first-passage problem, which results from conformation fluctuations of the motor switch, we calculated P(Δf) and P(Δb) and found good agreement with the measurements.

Bacterial chemotaxis in an optical trap.

An optical trapping technique is implemented to investigate the chemotactic behavior of a marine bacterial strain Vibrio alginolyticus. The technique takes the advantage that the bacterium has only a single polar flagellum, which can rotate either in the counter-clock-wise or clock-wise direction. The two rotation states of the motor can be readily and instantaneously resolved in the optical trap, allowing the flagellar motor switching rate S(t) to be measured under different chemical stimulations. In this paper the focus will be on the bacterial response to an impulsive change of chemoattractant serine. Despite different propulsion apparati and motility patterns, cells of V. alginolyticus apparently use a similar response as Escherichia coli to regulate their chemotactic behavior. Specifically, we found that the switching rate S(t) of the bacterial motor exhibits a biphasic behavior, showing a fast initial response followed by a slow relaxation to the steady-state switching rate S0. The measured S(t) can be mimicked by a model that has been recently proposed for chemotaxis in E. coli. The similarity in the response to the brief chemical stimulation in these two different bacteria is striking, suggesting that the biphasic response may be evolutionarily conserved. This study also demonstrated that optical tweezers can be a useful tool for chemotaxis studies and should be applicable to other polarly flagellated bacteria.