Summary Quantitative spatial distributions of ribosomes (S2-YFP) and RNA polymerase (β′-yGFP) in live E. 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, attributed to free mRNA being translated by one or more 70S ribosomes. We find no clear evidence of sub-diffusion, as would arise from tethering of ribosomes. 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.

Most biochemical and biophysical studies of the reaction mechanisms, kinetics, and thermodynamics of proteins and nucleic acids are carried out in vitro using dilute aqueous solutions of purified biopolymer constituents. However, the cytoplasm of both prokaryotic and eukaryotic cells contains a very high total concentration of proteins, nucleic acids, lipids, and supramolecular assemblies of these constituents. In Escherichia coli grown at moderate osmolality, the typical total mass density of protein and nucleic acid within the cytoplasm is ϳ220 mg/ml (5), distributed among the nucleoid, ribosomes, and a diverse collection of smaller proteins, tRNA, and mRNA (3). Taken together, these macromolecules occupy some 20 to 30% of the total cytoplasmic volume.In a crowded fluid, each molecule is excluded from much of the total volume by the presence of other biopolymers (11, 37). In thermodynamic terms, excluded volume decreases the translational entropy of each species, increases free energy, and, thus, increases the thermodynamic driving force to react or bind. These effects could be very large; the thermodynamic activity (the "effective concentration") of a typical globular protein could be Ͼ100 times higher in the E. coli cytoplasm than at the same concentration in an uncrowded solution (9). Crowding also dramatically affects diffusion (26), which is critical for normal cell function and growth (28). Excluded volume slows diffusion by making it less likely that a probe particle can find space in which to move without simultaneous, cooperative motion of several or many background particles (26).While the bacterial cytoplasm is often assumed to be a crowded aqueous solution (37), the physical state of the cytoplasm is uncertain. Particularly for the low water content induced by hyperosmotic stress, the cytoplasm might become a biopolymer meshwork comprising the nucleoid, associated proteins, nascent mRNA, ribosomes, polypeptide chains, and strongly associated water (13, 38). Such conditions are reminiscent of a polymeric hydrogel (1). Confinement within the pores of the meshwork would enhance protein binding equilibria and slow protein diffusion in a manner qualitatively similar to crowding.Strong effects of crowding and confinement in vitro have been observed for the tracer diffusion of globular proteins (26) and for the diffusion of tracer proteins in concentrated solutions of hydrophilic polymers (2, 8) and in hydrogels (1). The apparent diffusion coefficient decreases roughly exponentially with the macromolecular volume fraction , in agreement with a parametrized model called scaled particle theory (SPT) (14,26). Additional studies in vitro have shown crowding effects on protein folding (36) and association (24), on the thermodynamics of the protein-nucleic acid interactions critical to replication (27), on enzyme kinetics (25), and on the stability of protein oligomers such as F-actin (16, 20) and of fibrils such as ␤-amyloid (15).We know of no experimental studies of crowding/confinement effects on protein di...

For reactions of gas phase, ground state, neutral transition metal atoms from the 4d series with alkanes and alkenes, we combine 300 K kinetics measurements with ab initio electronic structure calculations to infer mechanisms in some detail. The theoretical method PCI-80 with zero-point energy corrections to the bare potential surface apparently produces bond energies, reaction exothermicities, and even saddle point energies accurate to within 2-3 kcal/mol, provided that the correct ground state has been located, which is sometimes difficult. The reactions fall into two general categories: termolecular stabilization of long-lived M(hydrocarbon) complexes and bimolecular elimination of Hz. By using the ab initio energies and vibrational frequencies in a statistical unimolecular rate theory (RRKM theory), we can model the lifetimes of M(hydrocarbon) complexes to assess the plausibility of a saturated termolecular mechanism at 1 Torr He. Termolecular examples include the reactions of Pd with alkanes to form long-range v2 complexes; the reactions of Rh and Pd with alkenes to form n complexes; and probably the reactions of Y, Zr, Nb, Rh, and Pd with cyclopropane to form CH or CC insertion complexes. In other reactions, all of the evidence indicates a bimolecular H2 elimination mechanism. Rhodium is unique among the 4d metal atoms in effecting HZ elimination from ethane and larger alkanes. Yttrium, zirconium, and niobium almost surely insert in CH bonds of ethylene and larger alkenes, ultimately eliminating H2. We discuss the general requirements on the pattern of atomic electronic states that pennit efficient CH bond activation and H2 elimination. The good agreement between the observed reaction rates and the PCI-80 calculations lends confidence to future efforts to apply ab initio techniques to more complicated catalytic systems, including condensed phase reactions involving ligated metal centers.

Natural antimicrobial peptides (AMPs) provide prototypes for the design of unconventional antimicrobial agents. Existing bulk assays measure AMP activity but do not provide details of the growthhalting mechanism. We use fluorescence microscopy to directly observe the attack of the human antimicrobial peptide LL-37 on single Escherichia coli cells in real time. Our findings strongly suggest that disruption of the cytoplasmic membrane is not the growth-halting mechanism. At 8 μM, LL-37 binding saturates the outer membrane (OM) within 1 min. Translocation across the OM and access to the periplasmic space (5-25 min later) correlates in time with the halting of growth. Septating cells are attacked more readily than nonseptating cells. The halting of growth may occur because of LL-37 interference with cell wall biogenesis. Only well after growth halts does the peptide permeabilize the cytoplasmic membrane to GFP and the small dye Sytox Green. The assay enables dissection of antimicrobial design criteria into two parts: translocation across the OM and the subsequent halting of growth.

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