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Biomedical subjects

Howard C Berg

Publications and source records attributed to Howard C Berg.

At least 19 recordsLinked to original sources
PubMed2006

Monitoring bacterial chemotaxis by using bioluminescence resonance energy transfer: absence of feedback from the flagellar motors.

We looked for a feedback system in Escherichia coli that might sense the rotational bias of flagellar motors and regulate the activity of the chemotaxis receptor kinase. Our search was based on the assumption that any machinery that senses rotational bias will be perturbed if flagellar rotation stops. We monitored the activity of the kinase in swimming cells by bioluminescence resonance energy transfer (BRET) between Renilla luciferase fused to the phosphatase, CheZ, and yellow fluorescent protein fused to the response regulator, CheY. Then we jammed the flagellar motors by adding an antifilament antibody that crosslinks adjacent filaments in flagellar bundles. At steady state, the rate of phosphorylation of CheY is equal to the rate of dephosphorylation of CheY-P, which is proportional to the degree of association between CheZ and CheY-P, the quantity sensed by BRET. No changes were observed, even upon addition of an amount of antibody that stopped the swimming of >95% of cells within a few seconds. So, the kinase does not appear to be sensitive to motor output. The BRET technique is complementary to one based on FRET, described previously. Its reliability was confirmed by measurements of the response of cells to the addition of attractants.

Antibodies, Bacterial↗
PubMed2006

Osmotic stress mechanically perturbs chemoreceptors in Escherichia coli.

Two-component signaling systems play a major role in the long-term adaptation of microorganisms to changes in osmolarity, but how osmoreceptors work is not well understood. Temporal changes in solute concentration are sensed by the chemotaxis system in Escherichia coli, enabling these bacteria to avoid regions of high osmolarity. To study how osmolarity is detected in this system, we fused yellow fluorescent protein (YFP) to the C terminus of the serine or aspartate chemoreceptor, monitored the steady-state fluorescence polarization of YFP, and found that the polarization decreased substantially upon addition of osmotic agents. This decrease was due to an increase in fluorescence resonance energy transfer between YFP fluorophores in adjacent homodimers within trimers of dimers. Thus, changes in homodimer spacing and/or orientation appear to initiate osmotactic signaling.

Escherichia coli K12↗
PubMed2005

Swarming motility: it better be wet.

When grown on a soft agar surface in a rich medium, cells of Salmonella typhimurium elongate, produce extra flagella and move over the surface in a coordinated manner. In mutants with defects in the chemotaxis signaling pathway, the agar plates remain dry and the cells' flagella are short. Recent work shows that the anti-sigma factor controlling late-gene flagellar synthesis is secreted less by flagella when things are dry: the flagellum senses wetness.

Bacterial Proteins↗
PubMed2005

Escherichia coli swim on the right-hand side.

The motion of peritrichously flagellated bacteria close to surfaces is relevant to understanding the early stages of biofilm formation and of pathogenic infection. This motion differs from the random-walk trajectories of cells in free solution. Individual Escherichia coli cells swim in clockwise, circular trajectories near planar glass surfaces. On a semi-solid agar substrate, cells differentiate into an elongated, hyperflagellated phenotype and migrate cooperatively over the surface, a phenomenon called swarming. We have developed a technique for observing isolated E. coli swarmer cells moving on an agar substrate and confined in shallow, oxidized poly(dimethylsiloxane) (PDMS) microchannels. Here we show that cells in these microchannels preferentially 'drive on the right', swimming preferentially along the right wall of the microchannel (viewed from behind the moving cell, with the agar on the bottom). We propose that when cells are confined between two interfaces--one an agar gel and the second PDMS--they swim closer to the agar surface than to the PDMS surface (and for much longer periods of time), leading to the preferential movement on the right of the microchannel. Thus, the choice of materials guides the motion of cells in microchannels.

Agar↗
PubMed2004

Single-cell FRET imaging of phosphatase activity in the Escherichia coli chemotaxis system.

Two-component signaling systems, in which a receptor-coupled kinase is used to control the phosphorylation level of a response regulator, are commonly used in bacteria to sense their environment. In the chemotaxis system of Escherichia coli, the receptors, and thus the kinase, are clustered on the inner cell membrane. The phosphatase of this system also is recruited to receptor clusters, but the reason for this association is not clear. By using FRET imaging of single cells, we show that in vivo the phosphatase activity is substantially larger at the cluster, indicating that the signaling source (the kinase) and the signaling sink (the phosphatase) tend to be located at the same place in the cell. When this association is disrupted, a gradient in the concentration of the phosphorylated response regulator appears, and the chemotactic response is degraded. Such colocalization is inevitable in systems in which the activity of the kinase and the phosphatase are produced by the same enzyme. Evidently, this design enables a more rapid and spatially uniform response.

Bacterial Proteins↗
PubMed2004

Functional interactions between receptors in bacterial chemotaxis.

Bacterial chemotaxis is a model system for signal transduction, noted for its relative simplicity, high sensitivity, wide dynamic range and robustness. Changes in ligand concentrations are sensed by a protein assembly consisting of transmembrane receptors, a coupling protein (CheW) and a histidine kinase (CheA). In Escherichia coli, these components are organized at the cell poles in tight clusters that contain several thousand copies of each protein. Here we studied the effects of variation in the composition of clusters on the activity of the kinase and its sensitivity to attractant stimuli, monitoring responses in vivo using fluorescence resonance energy transfer. Our results indicate that assemblies of bacterial chemoreceptors work in a highly cooperative manner, mimicking the behaviour of allosteric proteins. Conditions that favour steep responses to attractants in mutants with homogeneous receptor populations also enhance the sensitivity of the response in wild-type cells. This is consistent with a number of models that assume long-range cooperative interactions between receptors as a general mechanism for signal integration and amplification.

Bacterial Proteins↗
PubMed2004

Proteogenomic mapping as a complementary method to perform genome annotation.

The accelerated rate of genomic sequencing has led to an abundance of completely sequenced genomes. Annotation of the open reading frames (ORFs) (i.e., gene prediction) in these genomes is an important task and is most often performed computationally based on features in the nucleic acid sequence. Using recent advances in proteomics, we set out to predict the set of ORFs for an organism based principally on expressed protein-based evidence. Using a novel search strategy, we mapped peptides detected in a whole-cell lysate of Mycoplasma pneumoniae onto a genomic scaffold and extended these "hits" into ORFs bound by traditional genetic signals to generate a "proteogenomic map". We were able to generate an ORF model for M. pneumoniae strain FH using proteomic data with a high correlation to models based on sequence features. Ultimately, we detected over 81% of the genomically predicted ORFs in M. pneumoniae strain M129 (the originally sequenced strain). We were also able to detect several new ORFs not originally predicted by genomic methods, various N-terminal extensions, and some evidence that would suggest that certain predicted ORFs are bogus. Some of these differences may be a result of the strain analyzed but demonstrate the robustness of protein analysis across closely related genomes. This technique is a cost-effective means to add value to genome annotation, and a prerequisite for proteome quantitation and in vivo interaction measures.

Chromosome Mapping↗
PubMed2004

Moving fluid with bacterial carpets.

We activated a solid-fluid interface by attaching flagellated bacteria to a solid surface. We adsorbed swarmer cells of Serratia marcescens to polydimethylsiloxane or polystyrene. The cell bodies formed a densely packed monolayer while their flagella continued to rotate freely. Motion of the fluid close to an extended flat surface, visualized with tracer beads, was dramatically enhanced compared to the motion farther away. The tracer beads revealed complex ever-changing flow patterns, some linear (rivers), others rotational (whirlpools). Typical features of this flow were small (tens of micro m) and reasonably stable (many minutes). The surface performed active mixing equivalent to diffusion with a coefficient of 2 x 10(-7) cm(2)/s. We call these flat constructs "bacterial carpets". When attached to polystyrene beads or to fragments of polydimethylsiloxane, the bacteria generated both translation and rotation. We call these constructs "auto-mobile beads" or "auto-mobile chips". Given the size and strength of the flow patterns near the carpets, the motion must be generated by small numbers of coordinated flagella. We should be able to produce larger and longer-range effects by increasing coordination.

Biofilms↗
PubMed2004

The complete genome and proteome of Mycoplasma mobile.

Although often considered "minimal" organisms, mycoplasmas show a wide range of diversity with respect to host environment, phenotypic traits, and pathogenicity. Here we report the complete genomic sequence and proteogenomic map for the piscine mycoplasma Mycoplasma mobile, noted for its robust gliding motility. For the first time, proteomic data are used in the primary annotation of a new genome, providing validation of expression for many of the predicted proteins. Several novel features were discovered including a long repeating unit of DNA of approximately 2435 bp present in five complete copies that are shown to code for nearly identical yet uniquely expressed proteins. M. mobile has among the lowest DNA GC contents (24.9%) and most reduced set of tRNAs of any organism yet reported (28). Numerous instances of tandem duplication as well as lateral gene transfer are evident in the genome. The multiple available complete genome sequences for other motile and immotile mycoplasmas enabled us to use comparative genomic and phylogenetic methods to suggest several candidate genes that might be involved in motility. The results of these analyses leave open the possibility that gliding motility might have arisen independently more than once in the mycoplasma lineage.

Amino Acid Sequence↗
PubMed2004

Energetics of gliding motility in Mycoplasma mobile.

Mycoplasma mobile glides on surfaces at up to 7 microm/s by an unknown mechanism. We studied the energetics that power gliding by using a novel, growth medium-free system. We found that cells could glide in defined media if the glass substrate is preconditioned by exposure to horse serum. The active component that potentiates gliding is sensitive to proteinase K treatment. We used the defined medium system to test the effect of various inhibitors, ionophores, and poisons on motility of M. mobile. Valinomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), N,N'-dicyclohexylcarbodiimide, phenamil, amiloride, rifampin, and puromycin had no short-term effects on gliding. We also confirmed that we were able to modulate the membrane potential with valinomycin and FCCP by using a potential-sensitive dye. Shifting the pH likewise had no effect on motility. These results rule out the use of conventional ion motive forces to power gliding. Arsenate had a dramatic inhibitory effect on gliding, and both the speed and the fraction of cells moving tracked ATP levels. Sodium orthovanadate had a slight but significant inhibitory effect on gliding. Taken together, these results suggest that the motor system of M. mobile is likely an ATPase or is directly coupled to an ATPase.

Adenosine Triphosphate↗
PubMed2003

The speed of the flagellar rotary motor of Escherichia coli varies linearly with protonmotive force.

A protonmotive force (pmf) across the cell's inner membrane powers the flagellar rotary motor of Escherichia coli. Speed is known to be proportional to pmf when viscous loads are heavy. Here we show that speed also is proportional to pmf when viscous loads are light. Two motors on the same bacterium were monitored as the cell was slowly deenergized. The first motor rotated the entire cell body (a heavy load), while the second motor rotated a small latex bead (a light load). The first motor rotated slowly and provided a measure of the cell's pmf. The second motor rotated rapidly and was compared with the first, to give the speed-pmf relation for light loads. Experiments were done at 24.0 degrees C and 16.2 degrees C, with initial speeds indicating operation well into the high-speed, low-torque regime. Speed was found to be proportional to pmf over the entire (accessible) dynamic range (0-270 Hz). If the passage of a fixed number of protons carries the motor through each revolution, i.e., if the motor is tightly coupled, a linear speed-pmf relation is expected close to stall, where the work done against the viscous load matches the energy dissipated in proton flow. A linear relation is expected at high speeds if proton translocation is rate-limiting and involves multiple steps, a model that also applies to simple proton channels. The present work shows that a linear relation is true more generally, providing an additional constraint on possible motor mechanisms.

Biophysical Phenomena↗
PubMed2002

The rotary motor of bacterial flagella.

Flagellated bacteria, such as Escherichia coli, swim by rotating thin helical filaments, each driven at its base by a reversible rotary motor, powered by an ion flux. A motor is about 45 nm in diameter and is assembled from about 20 different kinds of parts. It develops maximum torque at stall but can spin several hundred Hz. Its direction of rotation is controlled by a sensory system that enables cells to accumulate in regions deemed more favorable. We know a great deal about motor structure, genetics, assembly, and function, but we do not really understand how it works. We need more crystal structures. All of this is reviewed, but the emphasis is on function.

Bacterial Physiological Phenomena↗
PubMed2002

Binding of the Escherichia coli response regulator CheY to its target measured in vivo by fluorescence resonance energy transfer.

In Escherichia coli chemotaxis, signaling depends on modulation of the level of phosphorylation of CheY, a small protein that couples receptors and flagellar motors. Working in vivo, we used fluorescence resonance energy transfer (FRET) to measure the interaction of CheY approximately P with its target, FliM. Binding of CheY approximately P to FliM was found to be much less cooperative than motor switching; however, under the conditions of our experiment, most of the FliM appeared to be in the cytoplasm. We studied signal processing times in the chemotaxis pathway by measuring the changes in CheY approximately P binding to FliM on flash release of caged chemoeffectors. Following sudden addition of attractant, the amount of CheY approximately P bound to FliM decayed exponentially with a rate constant of about 2 s(-1). Following sudden addition of repellent, FliM occupancy increased with a rate constant of about 20 s(-1). Using these data, we were able to construct a simple model for the chemotactic pathway and to estimate values of rate constants for several key reactions.

Bacterial Proteins↗
PubMed2002

Force and velocity of mycoplasma mobile gliding.

The effects of temperature and force on the gliding speed of Mycoplasma mobile were examined. Gliding speed increased linearly as a function of temperature from 0.46 microm/s at 11.5 degrees C to 4.0 microm/s at 36.5 degrees C. A polystyrene bead was attached to the tail of M. mobile using a polyclonal antibody raised against whole M. mobile cells. Cells attached to beads glided at the same speed as cells without beads. When liquid flow was applied in a flow chamber, cells reoriented and moved upstream with reduced speeds. Forces generated by cells at various gliding speeds were calculated by multiplying their estimated frictional drag coefficients with their velocities relative to the liquid. The gliding speed decreased linearly with force. At zero speed, the force measurements extrapolated to 26 pN at 22.5 and 27.5 degrees C. At zero force, the speed extrapolated to 2.3 and 3.3 microm/s at 22.5 and 27.5 degrees C, respectively--the same speeds as those observed for free gliding cells. Cells attached to beads were also trapped by an optical tweezer, and the stall force was measured to be 26 to 28 pN (17.5 to 27.5 degrees C). The gliding speed depended on temperature, but the maximum force did not, suggesting that the mechanism is composed of at least two steps, one that generates force and another that allows displacement. Other implications of these results are discussed.

Bacterial Physiological Phenomena↗