James C. Weisshaar

Superresolution imaging of ribosomes and RNA polymerase in live Escherichia coli cells

Quantitative spatial distributions of ribosomes (S2‐YFP) and RNA polymerase (RNAP; β′‐yGFP) in live Escherichia coli are measured by superresolution fluorescence microscopy. In moderate growth conditions, nucleoid–ribosome segregation is strong, and RNAP localizes to the nucleoid lobes. The mean copy numbers per cell are 4600 RNAPs and 55 000 ribosomes. Only 10–15% of the ribosomes lie within the densest part of the nucleoid lobes, and at most 4% of the RNAPs lie in the two ribosome‐rich endcaps. The predominant observed diffusion coefficient of ribosomes is Dribo = 0.04 µm2 s−1, attributed to free mRNA being translated by one or more 70S ribosomes. We find no clear evidence of subdiffusion, as would arise from tethering of ribosomes to the DNA. The degree of DNA–ribosome segregation strongly suggests that in E. coli most translation occurs on free mRNA transcripts that have diffused into the ribosome‐rich regions. Both RNAP and ribosome radial distributions extend to the cytoplasmic membrane, consistent with the transertion hypothesis. However, few if any RNAP copies lie near the membrane of the endcaps. This suggests that if transertion occurs, it exerts a direct radially expanding force on the nucleoid, but not a direct axially expanding force.

Crowding and Confinement Effects on Protein Diffusion In Vivo

The first in vivo measurements of a protein diffusion coefficient versus cytoplasmic biopolymer volume fraction are presented. Fluorescence recovery after photobleaching yields the effective diffusion coefficient on a 1-mum-length scale of green fluorescent protein within the cytoplasm of Escherichia coli grown in rich medium. Resuspension into hyperosmotic buffer lacking K+ and nutrients extracts cytoplasmic water, systematically increasing mean biopolymer volume fraction, , and thus the severity of possible crowding, binding, and confinement effects. For resuspension in isosmotic buffer (osmotic upshift, or Delta, of 0), the mean diffusion coefficient, , in cytoplasm (6.1 +/- 2.4 microm2 s(-1)) is only 0.07 of the in vitro value (87 microm2 s(-1)); the relative dispersion among cells, sigmaD/ (standard deviation, sigma(D), relative to the mean), is 0.39. Both and sigmaD/ remain remarkably constant over the range of Delta values of 0 to 0.28 osmolal. For a Delta value of > or =0.28 osmolal, formation of visible plasmolysis spaces (VPSs) coincides with the onset of a rapid decrease in by a factor of 380 over the range of Delta values of 0.28 to 0.70 osmolal and a substantial increase in sigmaD/. Individual values of D vary by a factor of 9 x 10(4) but correlate well with f(VPS), the fractional change in cytoplasmic volume on VPS formation. The analysis reveals two levels of dispersion in D among cells: moderate dispersion at low Delta values for cells lacking a VPS, perhaps related to variation in phi or biopolymer organization during the cell cycle, and stronger dispersion at high Delta values related to variation in f(VPS). Crowding effects alone cannot explain the data, nor do these data alone distinguish crowding from possible binding or confinement effects within a cytoplasmic meshwork.

Understanding barriers to internal rotation in substituted toluenes and their cations

Author Institution: Lawrence Berkeley Laboratory, M/S 2-300, 1 Cyclotron Road, Berkeley, CA 94720; University of Wisconsin-Madison, Madison, WI 53706-1396.

Lipid mixing and content release in single-vesicle, SNARE-driven fusion assay with 1-5 ms resolution.

A single-vesicle, fluorescence-based, SNARE-driven fusion assay enables simultaneous measurement of lipid mixing and content release with 5 ms/frame, or even 1 ms/frame, time resolution. The v-SNARE vesicles, labeled with lipid and content markers of different color, dock and fuse with a planar t-SNARE bilayer supported on glass. A narrow (<5 ms duration), intense spike of calcein fluorescence due to content release and dequenching coincides with inner-leaflet lipid mixing within 10 ms. The spike provides more sensitive detection of productive hemifusion events than do lipid labels alone. Consequently, many fast events previously thought to be prompt, full fusion events are now reclassified as productive hemifusion. Both full fusion and hemifusion occur with a time constant of 5-10 ms. At 60% phosphatidylethanolamine lipid composition, productive and dead-end hemifusion account for 65% of all fusion events. However, quantitative analysis shows that calcein is released into the space above the bilayer (vesicle bursting), rather than the thin aqueous space between the bilayer and glass. Evidently, at the instant of inner-leaflet mixing, flattening of the vesicle increases the internal pressure beyond the bursting point. This may be related to in vivo observations suggesting that membrane lysis often competes with membrane fusion.

Collisionless nonradiative decay rates of single rotational levels of S1 formaldehyde

Fluorescence lifetimes of single rotational levels of the lowest vibrational level of the first excited singlet state of H2CO and D2CO have been measured under collision‐free conditions following excitation by a pulsed dye laser. For H2CO, the lifetimes range from 66 ns to 4.2 μs with a median of about 160 ns. Individual lifetimes show no systematic variation with J′, K′, or Erot. K‐doublet levels split by as little as 8×10−4cm−1 in S1 are observed to have different lifetimes. The H2CO results are interpreted in terms of a sequential coupling model (S1→S0→continuum) in which the final states are those of the H2+CO dissociation continuum. For D2CO, the lifetimes vary between 5.5 and 8.1 μs and are nearly radiative lifetimes.

Cytoplasmic Protein Mobility in Osmotically Stressed Escherichia coli

Facile diffusion of globular proteins within a cytoplasm that is dense with biopolymers is essential to normal cellular biochemical activity and growth. Remarkably, Escherichia coli grows in minimal medium over a wide range of external osmolalities (0.03 to 1.8 osmol). The mean cytoplasmic biopolymer volume fraction ((phi)) for such adapted cells ranges from 0.16 at 0.10 osmol to 0.36 at 1.45 osmol. For cells grown at 0.28 osmol, a similar phi range is obtained by plasmolysis (sudden osmotic upshift) using NaCl or sucrose as the external osmolyte, after which the only available cellular response is passive loss of cytoplasmic water. Here we measure the effective axial diffusion coefficient of green fluorescent protein (D(GFP)) in the cytoplasm of E. coli cells as a function of (phi) for both plasmolyzed and adapted cells. For plasmolyzed cells, the median D(GFP) (D(GFP)(m)) decreases by a factor of 70 as (phi) increases from 0.16 to 0.33. In sharp contrast, for adapted cells, D(GFP)(m) decreases only by a factor of 2.1 as (phi) increases from 0.16 to 0.36. Clearly, GFP diffusion is not determined by (phi) alone. By comparison with quantitative models, we show that the data cannot be explained by crowding theory. We suggest possible underlying causes of this surprising effect and further experiments that will help choose among competing hypotheses. Recovery of the ability of proteins to diffuse in the cytoplasm after plasmolysis may well be a key determinant of the time scale of the recovery of growth.

Collisionless decay, vibrational relaxation, and intermediate case quenching of S1 formaldehyde

The decay of fluorescence from the 40 and 41 levels of the S1(A 1A2) state of H2CO and D2CO has been monitored as a function of pressure after selective, pulsed laser excitation. For D2CO, single exponential decays modified by 40↔41 energy transfer were observed over the entire pressure range 4×10−5–4 Torr. The zero pressure lifetimes τ0(40) =7.8±0.7 μs and τ0(41) =6.0±0.4 μs are probably the radiative lifetimes. The rate of 41→40 energy transfer in D2CO was found to be (9.6±0.4) ×10−10 cm3 molecule−1 s−1, about three times the gas kinetic rate. For H2CO at pressures above 0.1 Torr, fluorescence decays were also single exponentials modified by 40 ↔41 energy transfer. However, in the range 2×10−4–0.1 Torr, the decays of the individual 40 and 41 vibronic levels were typically biexponential. The zero pressure decay occurs on a timescale at least 20 times faster than the radiative lifetime of ∼5 μs. The Stern‐Volmer plots of τ−1 vs pressure give quenching rates between 2.2×10−9 and 6.5×10−9 cm3 molecule−1 s−...

Protein Diffusion in the Periplasm of E. coli under Osmotic Stress

The physical and mechanical properties of the cell envelope of Escherichia coli are poorly understood. We use fluorescence recovery after photobleaching to measure diffusion of periplasmic green fluorescent protein and probe the fluidity of the periplasm as a function of external osmotic conditions. For cells adapted to growth in complete medium at 0.14-1.02 Osm, the mean diffusion coefficient increases from 3.4 μm² s⁻¹ to 6.6 μm² s⁻¹ and the distribution of D(peri) broadens as growth osmolality increases. This is consistent with a net gain of water by the periplasm, decreasing its biopolymer volume fraction. This supports a model in which the turgor pressure drops primarily across the thin peptidoglycan layer while the cell actively maintains osmotic balance between periplasm and cytoplasm, thus avoiding a substantial pressure differential across the cytoplasmic membrane. After sudden hyperosmotic shock (plasmolysis), the cytoplasm loses water as the periplasm gains water. Accordingly, increases threefold. The fluorescence recovery after photobleaching is complete and homogeneous in all cases, but in minimal medium, the periplasm is evidently thicker at the cell tips. For the relevant geometries, Brownian dynamics simulations in model cytoplasmic and periplasmic volumes provide analytical formulae for extraction of accurate diffusion coefficients from readily measurable quantities.

State-to-state photoionization of VO : propensity for large, positive changes in rotational quantum number

State‐to‐state threshold photoionization cross sections from specific spin–rotation levels N’=7, J’=8.5, v’=3 of C 4∑− VO to specific levels N+J+ of X 3∑− VO+ show a remarkable propensity for large, positive ΔN. Observed transitions span the ranges ΔN=−5 to +7 and ΔJ=−5.5 to +4.5. The adiabatic ionization potential of VO is 7.2386±0.0004 eV. The mean bond length of v+=0, X 3∑− VO+ is 1.561±0.003 A.

ISOTOPE, ELECTRIC FIELD, AND VIBRATIONAL STATE DEPENDENCE OF SINGLE ROTATIONAL LEVEL LIFETIMES OF S1 FORMALDEHYDE

Additional single rovibronic level lifetimes of S1 H2CO and D2CO have been measured under collisionless conditions. The H2CO 41 lifetimes vary at least a factor of 150, from 20 nsec to 3.10 μsec. The observed D2CO 41 lifetimes fluctuate about ±20% around a mean value of 6.2 μsec, which is probably close to the pure radiative lifetime. In contrast, the observed D2CO 43 lifetimes vary from 1.09 to 2.46 μsec and the 2143 lifetimes vary from 212 nsec to 1.61 μsec. The onset of rotational state lifetime fluctuations in D2CO thus coincides with the high pressure D2+CO photochemical threshold. All of these results are explained in terms of a collisionless sequential decay mechanism, S1→S0→H2(D2)+CO. The last step probably involves tunneling through a barrier for the lower energies studied. For several H2CO 41 rotational levels application of a uniform external electric field of 0–4.6 kV/cm can change the fluorescence lifetime by at least a factor of 4. This result is understood in terms of small (≲0.05 cm−1) shi...