partly due to the methods of extracting the representative average values. Some authors take the arithmetic average [6,7,8]. Upon visual inspection of the data obtained for E.coli (Fig. 3), it is apparent that the median value may actually be a better approximation of the ‘average’ diffusion coefficient. This approach has been implemented in later studies [5,13]. Another possible reason for the differences in the diffusion coefficients is the composition of the growth media. It can be observed that cells grown in rich media like LB show lower mobility of proteins [6,7,13] than cells grown in minimal media [5]. Particularly striking are the values reported by the group of Weisshaar [5,6]. Using ‘‘conventional FRAP’’, they report diffusion coefficient for GFP [6] of 6–7 mm2/s for LB grown cells, whereas cells grown in a minimal MOPS-based medium showed values of 14 mm2/s [5]. Moreover, they reported that the culture’s history has a major impact on the mobility of GFP diffusion [5]. Cells adapted to high osmolality of growth tend to display a lower decrease in diffusion coefficients upon exposure to osmotic upshift than cells grown in media of normal osmolality. The observation that in bacteria grown in rich media the diffusion of macromolecules is slower than in bacteria grown in minimal medium may have a plausible biological rationale. Bacteria grown in rich media divide more often and synthesize more mRNA [15] and have a higher number of ribosomes [16] as compared to bacteria growing and dividing more slowly in minimal medium. In this context the overall macromolecular crowding (often thought to be the main cause for slower diffusion References 1. Elowitz MB, Surette MG, Wolf PE, Stock JB, Leibler S (1999) Protein mobility in the cytoplasm of Escherichia coli. J Bacteriol 181: 197–203. 2. Axelrod D, Koppel DE, Schlessinger J, Elson E, Webb WW (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J 16: 1055–1069. 3. Nenninger A, Mastroianni G, Mullineaux CW (2010) Size-dependence of protein diffusion in the cytoplasm of Escherichia coli. J Bacteriol. 4. Kumar M, Mommer MS, Sourjik V (2010) Mobility of cytoplasmic, membrane, and DNA-binding proteins in Escherichia coli. Biophys J 98: 552–559. 5. Konopka MC, Sochacki KA, Bratton BP, Shkel IA, Record MT, et al. (2009) Cytoplasmic protein mobility in osmotically stressed Escherichia coli. J Bacteriol 191: 231–237. 6. Konopka MC, Shkel IA, Cayley S, Record MT, Weisshaar JC (2006) Crowding and confinement effects on protein diffusion in vivo. J Bacteriol 188: 6115–6123. 7. van den Bogaart G, Hermans N, Krasnikov V, Poolman B (2007) Protein mobility and diffusive barriers in Escherichia coli: consequences of osmotic stress. Mol Microbiol 64: 858–871. 8. Slade KM, Baker R, Chua M, Thompson NL, Pielak GJ (2009) Effects of recombinant protein expression on green fluorescent protein diffusion in Escherichia coli. Biochemistry 48: 5083–5089. Evaluation of FRAP for D Measurements in Bacteria in the cytoplasm as compared to dilute aqueous solutions) may be rather constant, however, the nature of the crowding agent would change. It is tempting to speculate that in the cytoplasm of bacteria grown on rich media the diffusion is slower due to a higher abundance of ribosomes that are bulkier crowders (11.4 nm radius, [5]) than average cytoplasmatic proteins (2–3nm diameter). Moreover, the increased content of mRNA, which forms long unfolded chains, may additionally hinder macromolecule diffu- sion. The hypothesis that not only the cytoplasm crowding itself, but also the nature of the crowders contributes to slowed diffusion remains yet to be proven experimentally. Conclusions We have shown that, under a variety of osmotic conditions, pulsed-FRAP and ‘‘conventional-FRAP’’ yield very similar diffusion coefficients of GFP in the cytoplasm of E.coli. We speculate that the different values reported in the literature are due to variations in the constitution of the cells, i.e. as determined by their growth media and history. Author Contributions Conceived and designed the experiments: JTM VK BP. Performed the experiments: JTM VK. Analyzed the data: JTM VK. Contributed reagents/materials/analysis tools: VK GvdB FdH. Wrote the paper: JTM VK BP. 9. Slade KM, Steele BL, Pielak GJ, Thompson NL (2009) Quantifying green fluorescent protein diffusion in Escherichia coli by using continuous photo- bleaching with evanescent illumination. J Phys Chem B 113: 4837–4845. 10. English BP, Sanamrad A, Tankov S, Hauryliuk V, Elf J (2010) Tracking of individual freely diffusing fluorescent protein molecules in the bacterial cytoplasm. 11. Cluzel P, Surette M, Leibler S (2000) An ultrasensitive bacterial motor revealed by monitoring signaling proteins in single cells. Science 287: 1652–1655. 12. Mika JT, Poolman B (2011) Macromolecule diffusion and confinement in prokaryotic cells. Curr Opin Biotechnol 22: 117–126. 13. Mika JT, van den Bogaart G, Veenhoff L, Krasnikov V, Poolman B (2010) Molecular sieving properties of the cytoplasm of Escherichia coli and consequences of osmotic stress. Mol Microbiol. 14. Mullineaux CW, Nenninger A, Ray N, Robinson C (2006) Diffusion of green fluorescent protein in three cell environments in Escherichia coli. J Bacteriol 188: 3442–3448. 15. Vogel U, Jensen KF (1994) The RNA chain elongation rate in Escherichia coli depends on the growth rate. J Bacteriol 176: 2807–2813. 16. Dennis PP, Bremer H (1974) Macromolecular composition during steady-state growth of Escherichia coli B-r. J Bacteriol 119: 270–281. PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e25664