Bacteria are the simplest living organisms. In particular, Escherichia coli has been extensively studied and it has become one of the standard model systems in microbiology. However, optical microscopy studies of single E. coli have been limited by its small size, approximately 1 x 3 microm, not much larger than the optical resolution, approximately 0.25 microm. As a result, not enough quantitative dynamical information on the life cycle of single E. coli is presently available. We suggest that, by careful analysis of images from phase contrast and fluorescence time-lapse microscopy, this limitation can be bypassed. For example, we show that applying this approach to monitoring morphogenesis in individual E. coli leads to a simple, quantitative description of this process. First, we find the time when the formation of the septum starts, tau(c). It occurs much earlier than the time when the constriction can be directly observed by phase contrast. Second, we find that the growth law of single cells is more likely bilinear/trilinear than exponential. This is further supported by the relations that hold between the corresponding growth rates. These methods could be further extended to study the dynamics of cell components, e.g., the nucleoid and the Z-ring.

SummaryTo detect and characterize membrane domains that have been proposed to exist in bacteria, two kinds of pyrene-labelled phospholipids, 2-pyrene-decanoylphosphatidylethanolamine (PY-PE) and 2-pyrenedecanoyl-phosphatidylglycerol (PY-PG) were inserted into Escherichia coli or Bacillus subtilis membrane. The excimerization rate coefficient, calculated from the excimer-to-monomer ratio dependencies on the probe concentration, was two times higher for PY-PE than for PY-PG at 37 ∞ ∞ ∞ ∞ C. This was ascribed to different local concentrations rather than to differences in mobility. The extent of mixing between the two fluorescent phospholipids, estimated by formation of their heteroexcimer, was found very low both in E. coli and B. subtilis , in contrast to model membranes. In addition, these two pyrene derivatives exhibited different temperature phase transitions and different detergent extractability, indicating that the surroundings of these phospholipids in bacterial membrane differ in organization and order. Inhibition of protein synthesis, leading to condensation of nucleoid and presumably to dissipation of membrane domains, indeed resulted in increased formation of heteroexcimers, broadening of phase transitions and equal detergent extractability of both probes. It is proposed that in bacterial membranes these phospholipids are segregated into distinct domains that differ in composition, proteo-lipid interaction and degree of order; the proteo-lipid domain being enriched by PE.

We monitor the shape dynamics of individual E. coli cells using time-lapse microscopy together with accurate image analysis. This allows measuring the dynamics of single-cell parameters throughout the cell cycle. In previous work, we have used this approach to characterize the main features of single-cell morphogenesis between successive divisions. Here, we focus on the behavior of the parameters that are related to cell division and study their variation over a population of 30 cells. In particular, we show that the single-cell data for the constriction width dynamics collapse onto a unique curve following appropriate rescaling of the corresponding variables. This suggests the presence of an underlying time scale that determines the rate at which the cell cycle advances in each individual cell. For the case of cell length dynamics a similar rescaling of variables emphasizes the presence of a breakpoint in the growth rate at the time when division starts, tau(c). We also find that the tau(c) of individual cells is correlated with their generation time, tau(g), and inversely correlated with the corresponding length at birth, L(0). Moreover, the extent of the T-period, tau(g) - tau(c), is apparently independent of tau(g). The relations between tau(c), tau(g) and L(0) indicate possible compensation mechanisms that maintain cell length variability at about 10%. Similar behavior was observed for both fast-growing cells in a rich medium (LB) and for slower growth in a minimal medium (M9-glucose). To reveal the molecular mechanisms that lead to the observed organization of the cell cycle, we should further extend our approach to monitor the formation of the divisome.

In this study we sought the detection and characterization of bacterial membrane domains. Fluorescence generalized polarization (GP) spectra of laurdan-labeled Escherichia coli and temperature dependencies of both laurdan's GP and fluorescence anisotropy of 1,3-diphenyl-1,3,5-hexatriene (DPH) (rDPH) affirmed that at physiological temperatures, the E. coli membrane is in a liquid-crystalline phase. However, the strong excitation wavelength dependence of rlaurdan at 37 degrees C reflects membrane heterogeneity. Time-resolved fluorescence emission spectra, which display distinct biphasic redshift kinetics, verified the coexistence of two subpopulations of laurdan. In the initial phase, <50 ps, the redshift in the spectral mass center is much faster for laurdan excited at the blue edge (350 nm), whereas at longer time intervals, similar kinetics is observed upon excitation at either blue or red edge (400 nm). Excitation in the blue region selects laurdan molecules presumably located in a lipid domain in which fast intramolecular relaxation and low anisotropy characterize laurdan's emission. In the proteo-lipid domain, laurdan motion and conformation are restricted as exhibited by a slower relaxation rate, higher anisotropy and a lower GP value. Triple-Gaussian decomposition of laurdan emission spectra showed a sharp phase transition in the temperature dependence of individual components when excited in the blue but not in the red region. At least two kinds of domains of distinct polarity and order are suggested to coexist in the liquid-crystalline bacterial membrane: a lipid-enriched and a proteolipid domain. In bacteria with chloramphenicol (Cam)-inhibited protein synthesis, laurdan showed reduced polarity and restoration of an isoemissive point in the temperature-dependent spectra. These results suggest a decrease in membrane heterogeneity caused by Cam-induced domain dissipation.

Flagella and cilia play a critical role in eukaryotic cell motility. Among the most notable waveforms exhibited by eukaryotic flagella are planar and helical waves observed in mammalian sperm and protozoa. Here we report on a highspeed study of the flagellar motility of the protozoan parasite Trypanosoma brucei responsible for the African sleeping sickness whose vector is the tsetse fly. In this organism, the flagellum is physically attached along the length of the tapering cell body, unlike the case of mammalian sperm where the flagellum is attached to the body only at one attachment site. Earlier studies had reported that propulsion was driven by helical waves propagating from the flagellar tip to the base with left-handed helicity. Using a millisecond-timescale microscope, we discovered a novel form of eukaryotic cell motility, in which alternating left-handed and right-handed helical waves (termed ''bihelical waves'') propagate along the flagellum and are separated by a moving kink. These bihelical waves produce torsion in the cell body that is resolved by a rocking motion but -unlike the case of mammalian sperm or the existing model for T. bruceiwithout net rotation. We also observed the rapid motion of the flagellum tip, for which we recorded velocities up to 673 nm/ms, about 96 times greater than the velocity of dynein motors in vivo. The forward translational movement of the body is coupled to both the rocking of the posterior cell body about its own axis and the axis of locomotion as well as the propagation of the bihelical waves and kinks. Our results demonstrate that millisecond-timescale microscopy is essential for studies of cell locomotion in microorganisms.