Running and tumbling with E. coli in polymeric solutions.

Run-and-tumble motility is widely used by swimming microorganisms including numerous prokaryotic and eukaryotic organisms. Here, we experimentally investigate the run-and-tumble dynamics of the bacterium E. coli in polymeric solutions. We find that even small amounts of polymer in solution can drastically change E. coli dynamics: cells tumble less and their velocity increases, leading to an enhancement in cell translational diffusion and a sharp decline in rotational diffusion. We show that suppression of tumbling is due to fluid viscosity while the enhancement in swimming speed is mainly due to fluid elasticity. Visualization of single fluorescently labeled DNA polymers reveals that the flow generated by individual E. coli is sufficiently strong to stretch polymer molecules and induce elastic stresses in the fluid, which in turn can act on the cell in such a way to enhance its transport. Our results show that the transport and spread of chemotactic cells can be independently modified and controlled by the fluid material properties.

Fluctuations and rheology in active bacterial suspensions.

We probe nonequilibrium properties of an active bacterial bath through measurements of correlations of passive tracer particles and the response function of a driven, optically trapped tracer. These measurements demonstrate violation of the fluctuation-dissipation theorem and enable us to extract the power spectrum of the active stress fluctuations. In some cases, we observe 1/sqrt[omega] scaling in the noise spectrum which we show can be derived from a theoretical model incorporating coupled stress, orientation, and concentration fluctuations of the bacteria.

Tracking single proteins within cells.

We present experiments in which single proteins were imaged and tracked within mammalian cells. Single proteins of R-phycoerythrin (RPE) were imaged by epifluorescence microscopy in the nucleoplasm and cytoplasm at 71 frames/s. We acquired two-dimensional trajectories of proteins (corresponding to the projection of three-dimensional trajectories onto the plane of focus) for an average of 17 frames in the cytoplasm and 16 frames in the nucleus. Diffusion constants were determined from linear fits to the mean square displacement and from the mean displacement squared per frame. We find that the distribution of diffusion constants for RPE within cells is broader than the distributions obtained from RPE in a glycerol solution, from a Monte Carlo simulation, and from the theoretical distribution for simple diffusion. This suggests that on the time scales of our measurements, the motion of single RPE proteins in the cytoplasm and nucleoplasm cannot be modeled by simple diffusion with a unique diffusion constant. Our results demonstrate that it is possible to follow the motion of single proteins within cells and that the technique of single molecule tracking can be used to probe the dynamics of intracellular macromolecules.

Staphylococcus aureus RN6390 replicates and induces apoptosis in a pulmonary epithelial cell line.

Staphylococcus aureus frequently colonizes the airways of patients with compromised airway defenses (e.g., cystic fibrosis [CF] patients) for extended periods. Persistent and relapsing infections may be related to live S. aureus bacteria actively residing inside epithelial cells. In this study, we infected a respiratory epithelial cell line, which was derived from a CF patient, with S. aureus RN6390. Internalization of S. aureus was found to be time and dose dependent and could be blocked by cytochalasin D. Transmission electron microscopy revealed that internalized bacteria resided within endocytic vacuoles without any evidence of lysosomal fusion in a 24-h period. The results of internalization experiments and time-lapse fluorescence microscopy of epithelial cells infected with green fluorescent S. aureus indicate that, after an initial lag period of 7 to 9 h, intracellular bacteria began to replicate, with three to five divisions in a 24-h period, leading to apoptosis of infected cells. Induction of apoptosis required bacterial internalization and is associated with intracellular replication. The slow and gradual replication of S. aureus inside epithelial cells hints at the role of host factors or signals in bacterial growth and further suggests possible cross talk between host cells and S. aureus.

Imaging constitutive exocytosis with total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy has been applied to image the final stage of constitutive exocytosis, which is the fusion of single post-Golgi carriers with the plasma membrane. The use of a membrane protein tagged with green fluorescent protein allowed the kinetics of fusion to be followed with a time resolution of 30 frames/s. Quantitative analysis allowed carriers undergoing fusion to be easily distinguished from carriers moving perpendicularly to the plasma membrane. The flattening of the carriers into the plasma membrane is seen as a simultaneous rise in the total, peak, and width of the fluorescence intensity. The duration of this flattening process depends on the size of the carriers, distinguishing small spherical from large tubular carriers. The spread of the membrane protein into the plasma membrane upon fusion is diffusive. Mapping many fusion sites of a single cell reveals that there are no preferred sites for constitutive exocytosis in this system.

Energetics of inclusion-induced bilayer deformations.

The material properties of lipid bilayers can affect membrane protein function whenever conformational changes in the membrane-spanning proteins perturb the structure of the surrounding bilayer. This coupling between the protein and the bilayer arises from hydrophobic interactions between the protein and the bilayer. We analyze the free energy cost associated with a hydrophobic mismatch, i.e., a difference between the length of the protein's hydrophobic exterior surface and the average thickness of the bilayer's hydrophobic core, using a (liquid-crystal) elastic model of bilayer deformations. The free energy of the deformation is described as the sum of three contributions: compression-expansion, splay-distortion, and surface tension. When evaluating the interdependence among the energy components, one modulus renormalizes the other: e.g., a change in the compression-expansion modulus affects not only the compression-expansion energy but also the splay-distortion energy. The surface tension contribution always is negligible in thin solvent-free bilayers. When evaluating the energy per unit distance (away from the inclusion), the splay-distortion component dominates close to the bilayer/inclusion boundary, whereas the compression-expansion component is more prominent further away from the boundary. Despite this complexity, the bilayer deformation energy in many cases can be described by a linear spring formalism. The results show that, for a protein embedded in a membrane with an initial hydrophobic mismatch of only 1 A, an increase in hydrophobic mismatch to 1.3 A can increase the Boltzmann factor (the equilibrium distribution for protein conformation) 10-fold due to the elastic properties of the bilayer.

Gramicidin channel kinetics under tension.

We have measured the effect of tension on dimerization kinetics of the channel-forming peptide gramicidin A. By aspirating large unilamellar vesicles into a micropipette electrode, we are able to simultaneously monitor membrane tension and electrical activity. We find that the dimer formation rate increases by a factor of 5 as tension ranges from 0 to 4 dyn/cm. The dimer lifetime also increases with tension. This behavior is well described by a phenomenological model of membrane elasticity in which tension modulates the mismatch in thickness between the gramicidin dimer and membrane.

Self-assembly of membrane junctions.

We present a mechanism for the aggregation of mobile intermembrane junctions, such as the connexon dyad of gap junctions. The model demonstrates that intermembrane repulsion provides a powerful self-assembly pressure. If the membrane repulsion is strong enough to prevent membrane adhesion, then the self-assembly pressure is of effective infinite range.

Bone marrow fibrin ring granulomas and cytomegalovirus infection.

Fibrin ring granulomas of the bone marrow are described in two organ transplant patients (one renal, one cardiac) with disseminated cytomegalovirus infection. Infection was documented by viral cultures and seroconversion, and in both cases typical cytomegalic cells were identified in proximity to the fibrin ring granulomas. These represent the first case reports of bone marrow fibrin ring granulomas associated with cytomegalovirus.

Discrimination between mammalian RNases H-1 and H-2.

The two principal RNases H in mammalian cells, H-1 and H-2, differ in their responses to sale, divalent metal, and sulfhydryl inhibition. Specific reaction conditions that provide unambiguous discrimination between RNases H-1 and H-2 with only two assays are described. The assays were used for identification in a new purification procedure for RNases H-1 and H-2.

Discontinuous DNA synthesis by purified mammalian proteins.

Five proteins purified from mouse cells acting together efficiently convert a single-stranded circular DNA template to covalently closed duplex circle by a discontinuous mechanism. DNA polymerase alpha/primase with the assistance of alpha accessory factor covers the single-stranded circle with RNA-primed DNA fragments. Primers are removed by a combination of RNase H-1 and a 5'-exonuclease that was identified by its ability to complete this in vitro system. The 5'-exonuclease is required to remove residual one or two ribonucleotides at the primer/DNA junction that are resistant to RNase H-1. Gap filling is by the DNA polymerase alpha/primase, and DNA ligase I converts the DNA fragments to continuous strand. The concerted action of the five proteins emulates synthesis of the staging strand at the replication fork.

Two forms of DNA polymerase delta from mouse cells. Purification and properties.

A procedure is described for the purification from cultured mouse cells of two DNA polymerase "delta-like" enzymes, as defined by intrinsic 3'-exonuclease activity, inhibition by aphidicolin, and relative insensitivity to N2-(p-n-butylphenyl)-dGTP. One of the two enzymes has been purified to near homogeneity and, similar to the DNA polymerase delta from calf thymus described by Lee et al. (Lee, M. Y. W. T., Tan, C. K., Downey, K. M., and So, A. G. (1984) Biochemistry 23, 1906-1913), it has a total molecular mass of 178 kDa (from sedimentation velocity of 8.0 S and Stokes radius of 54 A) and is composed of one each of 125- and 50-kDa polypeptides. It also resembles the DNA polymerase delta of Lee et al. in being stimulated by proliferating cell nuclear antigen (PCNA). It is the first clear structural and functional counterpart of the calf thymus enzyme. The major difference between the mouse DNA polymerase delta and the calf thymus enzyme of Lee et al. is that, under specific conditions, the mouse enzyme is active with poly(dA).oligo(dT) in the absence of PCNA, whereas the activity of the calf thymus enzyme with this template is reported to be completely dependent on PCNA. The reason for this difference is not known at this time. The second mouse cell enzyme has a molecular mass of 112 kDa (from sedimentation velocity of 6.3 S and Stokes radius of 43.0 A) and consists of a single polypeptide of 123-125 kDa in denaturing gels (p125). On the basis of its apparent formation by dissociation of DNA polymerase delta, and multiple similarities with DNA polymerase delta in enzymatic properties, the p125 is provisionally identified as the 125-kDa polypeptide of DNA polymerase delta. The p125 does not respond to PCNA, suggesting that the 50-kDa polypeptide is required for the stimulation of DNA polymerase delta by PCNA. The presence of the p125 in cell extracts would explain reports that DNA polymerase delta consists of a single polypeptide of approximately 125 kDa and/or thast it has a smaller molecular mass than DNA polymerase delta of Lee et al. and is not affected by PCNA (this does not apply to PCNA-independent DNA polymerase delta-like enzymes with higher molecular mass than the polymerase delta of Lee et al., which have recently been named DNA polymerases epsilon).

An inhibitor of DNA polymerases alpha and delta in calf thymus DNA.

Most, although not all, samples of commercial calf thymus DNA were strongly inhibitory to DNA polymerase alpha; the inhibition made the DNA useless as a template for this enzyme. In a pre-assembled DNA polymerase assay mixture (minus enzyme but including activated DNA) the inhibition tended to diminish with time but at a rate that was not predictable, and some inhibition usually persisted. It was concluded that the inhibition was the result of contamination of the DNA by a heparin-like material on the basis of the following: 1) the inhibition could be reversed by treatment of the DNA with heparinase; 2) both the endogenous inhibitory effect of calf thymus DNA as well as the inhibitory effect of heparin on DNA polymerase alpha are reversed by protamine (which is known to prevent the antithrombin activity of heparin); 3) both the endogenous inhibition and inhibition by heparin are also reversed by ampholyte (which also prevents the antithrombin activity of heparin); and 4) both the endogenous and the heparin-induced inhibitory effects display the same spectrum of activity against mammalian DNA polymerases, i.e. both DNA polymerases alpha and delta are extremely sensitive whereas, DNA polymerases beta and gamma are resistant. The last result also suggests the use of heparin as a specific inhibitor of purified mammalian DNA polymerases alpha and delta, similar to the use of aphidicolin.

The mechanism of action of an accessory protein for DNA polymerase alpha/primase.

A previous paper reported the purification (from mouse cell extracts) and some of the properties of a protein, alpha accessory factor (AAF), that specifically stimulates DNA polymerase alpha/primase (1). We describe here studies on the mechanism of action of AAF. In the presence of AAF and a large excess of single-stranded circular DNA template, a molecule of DNA polymerase alpha/primase interacts with a single template DNA molecule priming and synthesizing multiple short DNA fragments covering thousands of nucleotides without detaching from the template, and, by many-fold repetition of the process, accomplishes serial replication of the population of DNA molecules. In contrast, without AAF the reaction involves the whole population of DNA molecules in parallel and with a very large number of binding events between DNA polymerase alpha/primase and DNA [corrected] template. The profound [corrected] increase in affinity of DNA polymerase alpha/primase for the DNA template that characterizes the mechanism suggests a functional identification of AAF as a template affinity protein. The resulting greater efficiency accounts for the ability of AAF to stimulate both the primase and polymerase activities of DNA polymerase alpha/primase. AAF also increases the processivity of DNA polymerase alpha/primase from approximately 15 to approximately 115 nucleotides, a size similar to that of mammalian Okazaki fragments, and it appears to allow DNA polymerase alpha/primase to traverse double-stranded regions of a DNA template. These features of the mechanism of AAF suggest that it may have a role in assisting DNA polymerase alpha/primase in synthesis of the lagging strand of a replication fork.