Cytoplasmic Protein Mobility in Osmotically Stressed
            <i>Escherichia coli</i>

Konopka, Michael; Sochacki, Kem A.; Bratton, Benjamin P.; Shkel, Irina A.; Record, M. Thomas; Weisshaar, James C.

doi:10.1128/jb.00536-08

Cited by 103 publications

(194 citation statements)

References 42 publications

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“…The signal sequence is cleaved from GFP on transport to the periplasm (19). For experiments on cytoplasmic GFP, we used the plasmid pMGS053, which expresses GFPmut2 using the lac promoter with repression maintained by pREP4 (Qiagen), as previously described (25). The WT MG1655 strain (lacking the plasmid) was used for several control experiments.…”

Section: Methodsmentioning

confidence: 99%

Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37

Sochacki

Barns²,

Bucki³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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236

272

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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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Section: Methodsmentioning

confidence: 99%

Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37

Sochacki

Barns²,

Bucki³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

236

272

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“…Clearly, biomacromolecular crowding in vivo somehow compensates for the ionic dependence of DNA-protein interactions in vitro, though how this happens from a physicochemical standpoint is not clear (37,174). Recent experiments showed that the growth rate and survival also depend on how fast the external osmolarity is raised; at low rates, cells can adjust and survive, but at high rates, cells are more likely to die (113).…”

Section: Biomacromolecular Crowdingmentioning

confidence: 99%

“…Although Brownian diffusion is absolutely necessary for cellular metabolism and signaling and appears generally fast enough in vivo, though slower than the case in vitro (13,48,59,116,140,234), particularly under osmotic stress (113,139,229), it is not sufficient for computerlike operation of the cell. Electrostatic potentials arising from transport of ions, modulated by covalently bound charges at biomacromolecular surfaces, must play a role (105), albeit one that is not well understood as yet, just as electrical potentials perform electronic computations in silicone-based semiconductors of integrated circuits.…”

Section: Zooming Out From Nanometers To Micrometersmentioning

confidence: 99%

“…Though chemical inhomogeneities of biomacromolecular surfaces provide an a priori physicochemical rationale for the model of supercrowded multiplexes, there is also experimental evidence that points to a similar concept: the relatively fast Brownian diffusion of various biomacromolecules and their GFP fusions within the cytoplasm of different cells and organelles (48,59,113,116,139,140,234). To rationalize such results, it has been suggested that mitochondria (highly crowded organelles) tend to supercrowd biomacromolecules mostly to the cytoplasmic side of the membrane, leaving the interior relatively empty for biomacromolecular diffusion (160).…”

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confidence: 99%

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From Water and Ions to Crowded Biomacromolecules:In VivoStructuring of a Prokaryotic Cell

Spitzer¹

2011

Microbiol Mol Biol Rev

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SUMMARY The interactions and processes which structure prokaryotic cytoplasm (water, ions, metabolites, and biomacromolecules) and ensure the fidelity of the cell cycle are reviewed from a physicochemical perspective. Recent spectroscopic and biological evidence shows that water has no active structuring role in the cytoplasm, an unnecessary notion still entertained in the literature; water acts only as a normal solvent and biochemical reactant. Subcellular structuring arises from localizations and interactions of biomacromolecules and from the growth and modifications of their surfaces by catalytic reactions. Biomacromolecular crowding is a fundamental physicochemical characteristic of cells in vivo . Though some biochemical and physiological effects of crowding (excluded volume effect) have been documented, crowding assays with polyglycols, dextrans, etc., do not properly mimic the compositional variety of biomacromolecules in vivo . In vitro crowding assays are now being designed with proteins, which better reflect biomacromolecular environments in vivo , allowing for hydrophobic bonding and screened electrostatic interactions. I elaborate further the concept of complex vectorial biochemistry, where crowded biomacromolecules structure the cytosol into electrolyte pathways and nanopools that electrochemically “wire” the cell. Noncovalent attractions between biomacromolecules transiently supercrowd biomacromolecules into vectorial, semiconducting multiplexes with a high (35 to 95%)-volume fraction of biomacromolecules; consequently, reservoirs of less crowded cytosol appear in order to maintain the experimental average crowding of ∼25% volume fraction. This nonuniform crowding model allows for fast diffusion of biomacromolecules in the uncrowded cytosolic reservoirs, while the supercrowded vectorial multiplexes conserve the remarkable repeatability of the cell cycle by preventing confusing cross talk of concurrent biochemical reactions.

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“…It points toward a key role molecular crowding plays in cell growth. The link between crowding and cell growth may be complex (37), as increased crowding slows diffusion (38), but also reduces unbinding (39) and effectively increases concentrations by reducing the accessible volume. However, if translation remains close to the diffusion limit, as we suggest here, slow diffusion should be the dominant effect of crowding (40,41).…”

Section: Coregulation Of Ribosomal and Trna-affiliated Proteins Corrementioning

confidence: 99%

Molecular crowding limits translation and cell growth

Klumpp

Scott

Pedersen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

268

359

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Bacterial growth is crucially dependent on protein synthesis and thus on the cellular abundance of ribosomes and related proteins. Here, we show that the slow diffusion of the bulky tRNA complexes in the crowded cytoplasm imposes a physical limit on the speed of translation, which ultimately limits the rate of cell growth. To study the required allocation of ancillary translational proteins to alleviate the effect of molecular crowding, we develop a model for cell growth based on a coarse-grained partitioning of the proteome. We find that coregulation of ribosome-and tRNA-affiliated proteins is consistent with measured growth-rate dependencies and results in near-optimal allocation over a broad range of growth rates. The analysis further resolves a long-standing controversy in bacterial growth physiology concerning the growth-rate dependence of translation speed and serves as a caution against premature identification of phenomenological parameters with mechanistic processes. Bacterial cell growth and protein synthesis are tightly coupled as proteins account for a large fraction of the cellular biomass (1). In the model organism Escherichia coli, over half of the biomass is protein (2), and protein synthesis accounts for more than two-thirds of the cell's ATP budget during rapid growth (3). Therefore, the machinery of protein synthesis, i.e., ribosomes, tRNAs, and ribosome-affiliated factors, plays a central role in maintaining exponential growth (1, 4). This is manifested by an increased ribosome content in rapidly growing cells (2,5,6), by direct observations that protein synthesis is limited by the availability of free ribosomes (7), and by considerations that link evolutionary selective pressure to the cost of protein synthesis (8).The most striking evidence for the central role of ribosomes in cell growth is provided by the linear relation between the ribosome mass fraction and the growth rate for bacteria grown in media containing different nutrients. This linear relation, which emerged from the systematic characterization of bacterial cells growing at different rates (5, 9), is illustrated in Fig. 1A with data for E. coli (2, 10, 11). It can be interpreted as reflecting the intrinsically autocatalytic activity of ribosomes synthesizing ribosomal proteins (9, 12) and identifies the fraction of ribosomes allocated to making ribosomal proteins as a key determinant of the growth rate (11). The picture that emerges from such considerations has formed the basis of a systematic theory of bacterial growth, based on empirical "growth laws", similar to the phenomenological laws of physics (11,13). The theory provides a successful framework for the analysis of the interdependence of cell growth and gene expression, of the effects of antibiotics, and of protein overexpression (11) without the need to characterize how the individual steps of synthesis and degradation are affected by the global state of the cell (14).In addition to their high ribosome content, rapidly growing bacteria also contain large amounts of othe...

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Cytoplasmic Protein Mobility in Osmotically Stressed Escherichia coli

Cited by 103 publications

References 42 publications

Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37

Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37

From Water and Ions to Crowded Biomacromolecules:In VivoStructuring of a Prokaryotic Cell

Molecular crowding limits translation and cell growth

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