DNA surface exploration and operator bypassing during target search.

Many proteins that bind specific DNA sequences search the genome by combining three-dimensional diffusion with one-dimensional sliding on nonspecific DNA1-5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore the DNA surface during the one-dimensional phase of target search. To track the rotation of sliding LacI molecules on the microsecond timescale, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluctuations in fluorescence signal are accurately described by rotation-coupled sliding, in which LacI traverses about 40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA; this suggests that the sliding protein frequently hops out of the DNA groove, which would result in the frequent bypassing of target sequences. We directly observe such bypassing using single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI hops one or two grooves (10-20 bp) every 200-700 µs. Our data suggest a trade-off between speed and accuracy during sliding: the weak nature of nonspecific protein-DNA interactions underlies operator bypassing, but also speeds up sliding. We anticipate that SMCT-FCS, which monitors rotational diffusion on the microsecond timescale while tracking individual molecules with millisecond resolution, will be applicable to the real-time investigation of many other biological interactions and will effectively extend the accessible time regime for observing these interactions by two orders of magnitude.

Bladderworts, the smallest known suction feeders, generate inertia-dominated flows to capture prey.

Aquatic bladderworts (Utricularia gibba and U. australis) capture zooplankton in mechanically triggered underwater traps. With characteristic dimensions less than 1 mm, the trapping structures are among the smallest known to capture prey by suction, a mechanism that is not effective in the creeping-flow regime where viscous forces prevent the generation of fast and energy-efficient suction flows. To understand what makes suction feeding possible on the small scale of bladderwort traps, we characterised their suction flows experimentally (using particle image velocimetry) and mathematically (using computational fluid dynamics and analytical mathematical models). We show that bladderwort traps avoid the adverse effects of creeping flow by generating strong, fast-onset suction pressures. Our findings suggest that traps use three morphological adaptations: the trap walls' fast release of elastic energy ensures strong and constant suction pressure; the trap door's fast opening ensures effectively instantaneous onset of suction; the short channel leading into the trap ensures undeveloped flow, which maintains a wide effective channel diameter. Bladderwort traps generate much stronger suction flows than larval fish with similar gape sizes because of the traps' considerably stronger suction pressures. However, bladderworts' ability to generate strong suction flows comes at considerable energetic expense.

Variation in Mutational Robustness between Different Proteins and the Predictability of Fitness Effects.

Random mutations in genes from disparate protein classes may have different distributions of fitness effects (DFEs) depending on different structural, functional, and evolutionary constraints. We measured the fitness effects of 156 single mutations in the genes encoding AraC (transcription factor), AraD (enzyme), and AraE (transporter) used for bacterial growth on l-arabinose. Despite their different molecular functions these genes all had bimodal DFEs with most mutations either being neutral or strongly deleterious, providing a general expectation for the DFE. This contrasts with the unimodal DFEs previously obtained for ribosomal protein genes where most mutations were slightly deleterious. Based on theoretical considerations, we suggest that the 33-fold higher average mutational robustness of ribosomal proteins is due to stronger selection for reduced costs of translational and transcriptional errors. Whereas the large majority of synonymous mutations were deleterious for ribosomal proteins genes, no fitness effects could be detected for the AraCDE genes. Four mutations in AraC and AraE increased fitness, suggesting that slightly advantageous mutations make up a significant fraction of the DFE, but that they often escape detection due to the limited sensitivity of commonly used fitness assays. We show that the fitness effects of amino acid substitutions can be predicted based on evolutionary conservation, but those weakly deleterious mutations are less reliably detected. This suggests that large-effect mutations and the fraction of highly deleterious mutations can be computationally predicted, but that experiments are required to characterize the DFE close to neutrality, where many mutations ultimately fixed in a population will occur.

Quantification of transcription factor-DNA binding affinity in a living cell.

The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [(3)H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 µM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 µM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element.

What matters for lac repressor search in vivo--sliding, hopping, intersegment transfer, crowding on DNA or recognition?

We have investigated which aspects of transcription factor DNA interactions are most important to account for the recent in vivo search time measurements for the dimeric lac repressor. We find the best agreement for a sliding model where non-specific binding to DNA is improbable at first contact and the sliding LacI protein binds at high probability when reaching the specific Osym operator. We also find that the contribution of hopping to the overall search speed is negligible although physically unavoidable. The parameters that give the best fit reveal sliding distances, including hopping, close to what has been proposed in the past, i.e. ∼40 bp, but with an unexpectedly high 1D diffusion constant on non-specific DNA sequences. Including a mechanism of inter-segment transfer between distant DNA segments does not bring down the 1D diffusion to the expected fraction of the in vitro value. This suggests a mechanism where transcription factors can slide less hindered in vivo than what is given by a simple viscosity scaling argument or that a modification of the model is needed. For example, the estimated diffusion rate constant would be consistent with the expectation if parts of the chromosome, away from the operator site, were inaccessible for searching.

Coverage-dependent adsorption and desorption of oxygen on Pd(100).

We have studied the adsorption and desorption of O2 on Pd(100) by supersonic molecular beam techniques and thermal desorption spectroscopy. Adsorption measurements on the bare surface confirm that O2 initially dissociates for all kinetic energies between 56 and 380 meV and surface temperatures between 100 and 600 K via a direct mechanism. At and below 150 K, continued adsorption leads to a combined O/O2 overlayer. Dissociation of molecularly bound O2 during a subsequent temperature ramp leads to unexpected high atomic oxygen coverages, which are also obtained at high incident energy and high surface temperature. At intermediate temperatures and energies, these high final coverages are not obtained. Our results show that kinetic energy of the gas phase reactant and reaction energy dissipated during O2 dissociation on the cold surface both enable activated nucleation of high-coverage surface structures. We suggest that excitation of local substrate phonons may play a crucial role in oxygen dissociation at any coverage.

Transcription-factor binding and sliding on DNA studied using micro- and macroscopic models.

Transcription factors search for specific operator sequences by alternating rounds of 3D diffusion with rounds of 1D diffusion (sliding) along the DNA. The details of such sliding have largely been beyond direct experimental observation. For this purpose we devised an analytical formulation of umbrella sampling along a helical coordinate, and from extensive and fully atomistic simulations we quantified the free-energy landscapes that underlie the sliding dynamics and dissociation kinetics for the LacI dimer. The resulting potential of mean force distributions show a fine structure with an amplitude of 1 k(B)T for sliding and 12 k(B)T for dissociation. Based on the free-energy calculations the repressor slides in close contact with DNA for 8 bp on average before making a microscopic dissociation. By combining the microscopic molecular-dynamics calculations with Brownian simulation including rotational diffusion from the microscopically dissociated state we estimate a macroscopic residence time of 48 ms at the same DNA segment and an in vitro sliding distance of 240 bp. The sliding distance is in agreement with previous in vitro sliding-length estimates. The in vitro prediction for the macroscopic residence time also compares favorably to what we measure by single-molecule imaging of nonspecifically bound fluorescently labeled LacI in living cells. The investigation adds to our understanding of transcription-factor search kinetics and connects the macro-/mesoscopic rate constants to the microscopic dynamics.

High fitness costs and instability of gene duplications reduce rates of evolution of new genes by duplication-divergence mechanisms.

An important mechanism for generation of new genes is by duplication-divergence of existing genes. Duplication-divergence includes several different submodels, such as subfunctionalization where after accumulation of neutral mutations the original function is distributed between two partially functional and complementary genes, and neofunctionalization where a new function evolves in one of the duplicated copies while the old function is maintained in another copy. The likelihood of these mechanisms depends on the longevity of the duplicated state, which in turn depends on the fitness cost and genetic stability of the duplications. Here, we determined the fitness cost and stability of defined gene duplications/amplifications on a low copy number plasmid. Our experimental results show that the costs of carrying extra gene copies are substantial and that each additional kilo base pairs of DNA reduces fitness by approximately 0.15%. Furthermore, gene amplifications are highly unstable and rapidly segregate to lower copy numbers in absence of selection. Mathematical modeling shows that the fitness costs and instability strongly reduces the likelihood of both sub- and neofunctionalization, but that these effects can be offset by positive selection for novel beneficial functions.

Lost in presumption: stochastic reactions in spatial models.

Physical modeling is increasingly important for generating insights into intracellular processes. We describe situations in which combined spatial and stochastic aspects of chemical reactions are needed to capture the relevant dynamics of biochemical systems.

Selection-driven gene loss in bacteria.

Gene loss by deletion is a common evolutionary process in bacteria, as exemplified by bacteria with small genomes that have evolved from bacteria with larger genomes by reductive processes. The driving force(s) for genome reduction remains unclear, and here we examined the hypothesis that gene loss is selected because carriage of superfluous genes confers a fitness cost to the bacterium. In the bacterium Salmonella enterica, we measured deletion rates at 11 chromosomal positions and the fitness effects of several spontaneous deletions. Deletion rates varied over 200-fold between different regions with the replication terminus region showing the highest rates. Approximately 25% of the examined deletions caused an increase in fitness under one or several growth conditions, and after serial passage of wild-type bacteria in rich medium for 1,000 generations we observed fixation of deletions that substantially increased bacterial fitness when reconstructed in a non-evolved bacterium. These results suggest that selection could be a significant driver of gene loss and reductive genome evolution.

Genomic buffering mitigates the effects of deleterious mutations in bacteria.

The relationship between the number of randomly accumulated mutations in a genome and fitness is a key parameter in evolutionary biology. Mutations may interact such that their combined effect on fitness is additive (no epistasis), reinforced (synergistic epistasis) or mitigated (antagonistic epistasis). We measured the decrease in fitness caused by increasing mutation number in the bacterium Salmonella typhimurium using a regulated, error-prone DNA polymerase (polymerase IV, DinB). As mutations accumulated, fitness costs increased at a diminishing rate. This suggests that random mutations interact such that their combined effect on fitness is mitigated and that the genome is buffered against the fitness reduction caused by accumulated mutations. Levels of the heat shock chaperones DnaK and GroEL increased in lineages that had accumulated many mutations, and experimental overproduction of GroEL further increased the fitness of lineages containing deleterious mutations. These findings suggest that overexpression of chaperones contributes to antagonistic epistasis.

Desorption of water from distinct step types on a curved silver crystal.

We have investigated the adsorption of H2O onto the A and B type steps on an Ag single crystal by temperature programmed desorption. For this study, we have used a curved crystal exposing a continuous range of surface structures ranging from [5(111) × (100)] via (111) to [5(111) × (110)]. LEED and STM studies verify that the curvature of our sample results predominantly from monoatomic steps. The sample thus provides a continuous array of step densities for both step types. Desorption probed by spatially-resolved TPD of multilayers of H2O shows no dependence on the exact substrate structure and thus confirms the absence of thermal gradients during temperature ramps. In the submonolayer regime, we observe a small and linear dependence of the desorption temperature on the A and B step density. We argue that such small differences are only observable by means of a single curved crystal, which thus establishes new experimental benchmarks for theoretical calculation of chemically accurate binding energies. We propose an origin of the observed behavior based on a "two state" desorption model.

Selection of resistant bacteria at very low antibiotic concentrations.

The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability to treat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically, but what role the much lower antibiotic concentrations present in many environments plays in selection remains largely unclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs at extremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to several hundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, even when present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations of antibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptible population. These results add another dimension to the evolution of resistance and suggest that the low antibiotic concentrations found in many natural environments are important for enrichment and maintenance of resistance in bacterial populations.

Stochastic reaction-diffusion kinetics in the microscopic limit.

Quantitative analysis of biochemical networks often requires consideration of both spatial and stochastic aspects of chemical processes. Despite significant progress in the field, it is still computationally prohibitive to simulate systems involving many reactants or complex geometries using a microscopic framework that includes the finest length and time scales of diffusion-limited molecular interactions. For this reason, spatially or temporally discretized simulations schemes are commonly used when modeling intracellular reaction networks. The challenge in defining such coarse-grained models is to calculate the correct probabilities of reaction given the microscopic parameters and the uncertainty in the molecular positions introduced by the spatial or temporal discretization. In this paper we have solved this problem for the spatially discretized Reaction-Diffusion Master Equation; this enables a seamless and physically consistent transition from the microscopic to the macroscopic frameworks of reaction-diffusion kinetics. We exemplify the use of the methods by showing that a phosphorylation-dephosphorylation motif, commonly observed in eukaryotic signaling pathways, is predicted to display fluctuations that depend on the geometry of the system.

Characterization of CO Adsorbed to Clean and Partially Oxidized Cu(211) and Cu(111).

Copper-based catalysts gain activity through the presence of poorly coordinated Cu atoms and incomplete oxidation at the surface. The catalytic mechanisms can in principle be observed by controlled dosing of reactants to single-crystal substrates. However, the interconnected influences of surface defects, partial oxidation, and adsorbate coverage present a large matrix of conditions that have not been fully explored in the literature. We recently characterized oxygen and carbon monoxide coadsorption on Cu(111), a nominally defect-free surface, and now extend our study to the stepped surface Cu(211). Temperature-programmed desorption of CO adsorbed to bare metal surfaces confirms that two sites dominate desorption from a saturated layer: atop terrace atoms of local (111) character and atop step edge atoms with CO bound more strongly to the latter. At low coverage, discrete CO resonances in reflection adsorption infrared spectra can be assigned to these sites: 2077 cm-1 for extended (111) terraces, 2093 cm-1 for step sites, and additional kink-adsorbed molecules at 2110 cm-1. With increasing coverage, in contrast to Cu(111), the infrared spectral features on Cu(211) evolve and shift as a consequence of dipole-dipole coupling between differentially occupied types of sites. Auger electron spectroscopy shows that exposure to background O2 oxidizes the (211) surface at a rate nearly 1 order of magnitude greater than (111); we argue that the resulting surface is stoichiometric Cu2O, as previously found for Cu(111). This oxide binds CO less strongly than the bare metal and the underlying crystal cut continues to influence the adsorption sites available to CO. On oxidized (111) terraces, broad absorption peaks at 2115-2120 cm-1; on oxidized Cu(211), CO adsorbed to step sites appears as a resolved secondary peak at 2144 cm-1. This suite of spectroscopic signatures, obtained under carefully controlled conditions, will help to determine the origin and fate of adsorbed species in future studies of reaction mechanisms on copper.

Eclipse period of R1 plasmids during downshift from elevated copy number: Nonrandom selection of copies for replication.

The classical Meselson-Stahl density-shift method was used to study replication of pOU71, a runaway-replication derivative of plasmid R1 in Escherichia coli. The miniplasmid maintained the normal low copy number of R1 during steady growth at 30°C, but as growth temperatures were raised above 34°C, the copy number of the plasmid increased to higher levels, and at 42°C, it replicated without control in a runaway replication mode with lethal consequences for the host. The eclipse periods (minimum time between successive replication of the same DNA) of the plasmid shortened with rising copy numbers at increasing growth temperatures (Olsson et al., 2003). In this work, eclipse periods were measured during downshifts in copy number of pOU71 after it had replicated at 39 and 42°C, resulting in 7- and 50-fold higher than normal plasmid copy number per cell, respectively. Eclipse periods for plasmid replication, measured during copy number downshift, suggested that plasmid R1, normally selected randomly for replication, showed a bias such that a newly replicated DNA had a higher probability of replication compared to the bulk of the R1 population. However, even the unexpected nonrandom replication followed the copy number kinetics such that every generation, the plasmids underwent the normal inherited number of replication, n, independent of the actual number of plasmid copies in a newborn cell.

Employing a cylindrical single crystal in gas-surface dynamics.

We describe the use of a polished, hollow cylindrical nickel single crystal to study effects of step edges on adsorption and desorption of gas phase molecules. The crystal is held in an ultra-high vacuum apparatus by a crystal holder that provides axial rotation about a [100] direction, and a crystal temperature range of 89 to 1100 K. A microchannel plate-based low energy electron diffraction/retarding field Auger electron spectrometer (AES) apparatus identifies surface structures present on the outer surface of the cylinder, while a separate double pass cylindrical mirror analyzer AES verifies surface cleanliness. A supersonic molecular beam, skimmed by a rectangular slot, impinges molecules on a narrow longitudinal strip of the surface. Here, we use the King and Wells technique to demonstrate how surface structure influences the dissociation probability of deuterium at various kinetic energies. Finally, we introduce spatially-resolved temperature programmed desorption from areas exposed to the supersonic molecular beam to show how surface structures influence desorption features.

RAIRS Characterization of CO and O Coadsorption on Cu(111).

In a study preliminary to investigating CO2 dissociation, we report our results on oxygen and carbon monoxide coadsorption on Cu(111). We use reflection adsorption infrared spectroscopy and Auger electron spectroscopy to characterize and quantify adsorbed species. On clean Cu(111), the CO internal stretch mode appears initially at 2077 cm-1 for a surface temperature of ∼80 K. We accurately reproduce the previously determined redshift of the absorption band with increasing CO coverage. We subsequently oxidize the surface by exposure to O2 at 300 K to ensure O2 dissociation. The band's frequency and line shape of subsequently adsorbed CO at ∼80 K are not affected. However, the maximum absorbance and integrated peak intensities drop with increasing O coverage. The data suggest that CO is not adsorbed near O, likely as a consequence of the mechanism of Cu(111) surface oxidation by O2 at 300 K. We discuss whether our RAIRS results may be used to quantify CO2 dissociation in the zero-coverage limit.

Ligand responses of Vfr, the virulence factor regulator from Pseudomonas aeruginosa.

Vfr, a transcription factor homologous to the Escherichia coli cyclic AMP (cAMP) receptor protein (CRP), regulates many aspects of virulence in Pseudomonas aeruginosa. Vfr, like CRP, binds to cAMP and then recognizes its target DNA and activates transcription. Here we report that Vfr has important functional differences from CRP in terms of ligand sensing and response. First, Vfr has a significantly higher cAMP affinity than does CRP, which might explain the mysteriously unidirectional functional complementation between the two proteins (S. E. H. West et al., J. Bacteriol. 176:7532-7542, 1994). Second, Vfr is activated by both cAMP and cGMP, while CRP is specific to cAMP. Mutagenic analyses show that Thr133 (analogous to Ser128 of CRP) is the key residue for both of these distinct Vfr properties. On the other hand, substitutions that cause cAMP-independent activity in Vfr are similar to those seen in CRP, suggesting that a common cAMP activation mechanism is present. In the course of these analyses, we found a remarkable class of Vfr variants that have completely reversed the regulatory logic of the protein: they are active in DNA binding without cAMP and are strongly inhibited by cAMP. The physiological impact of Vfr's ligand sensing and response is discussed, as is a plausible basis for the fundamental change in protein allostery in the novel group of Vfr variants.

Compensatory gene amplification restores fitness after inter-species gene replacements.

Genes introduced by gene replacements and other types of horizontal gene transfer (HGT) represent a significant presence in many archaeal and eubacterial genomes. Most alien genes are likely to be neutral or deleterious upon arrival and their long-term persistence may require a mechanism that improves their selective contribution. To examine the fate of inter-species gene replacements, we exchanged three native S. typhimurium genes encoding ribosomal proteins with orthologues from various other microbes. The results show that replacement of each of these three genes reduces fitness to such an extent that it would provide an effective barrier against inter-species gene replacements in eubacterial populations. However, these fitness defects could be partially ameliorated by gene amplification that augmented the dosage of the heterologous proteins. This suggests that suboptimal expression is a common fitness constraint for inter-species gene replacements, with fitness costs conferred by either a lower expression level of the alien protein compared with the native protein or a requirement for an increased amount of the alien protein to maintain proper function. Our findings can explain the observation that duplicated genes are over-represented among horizontally transferred genes, and suggest a potential coupling between compensatory gene amplification after HGT and the evolution of new genes.