fluorescence ͉ microscopy ͉ virus ͉ genome T he transfer of bacteriophage DNA from a capsid into the host cell is an event of great importance to biology and physics. In biology, DNA ejection was a key piece of evidence demonstrating that the genetic material was DNA and not protein (1), phages have long been used to insert foreign genes into bacteria (2), and phage-mediated DNA transfer between species is a challenge to theories of evolution (3). In physics, the translocation of DNA through a pore has been studied from the theoretical and experimental points of view (4-8). Because phage DNA ejection is such a well known example of this process, it is important to understand it from a quantitative point of view. This paper addresses a longstanding, quantitative puzzle about phage DNA ejection: How fast is the ejection process? We use bacteriophage , a typical tailed phage, to answer this question. In a infection, first the phage tail binds to the Escherichia coli outer membrane protein LamB, triggering ejection. Then the genome, 48.5 kbp of double-stranded DNA, moves out of the phage head, through the tail, and into the cytoplasmic space, which requires force on the DNA directed into the cell. A force of tens of piconewtons (pN) is produced by the highly bent and compressed DNA within the capsid (9-11), but not much is known about how fast the DNA transfer occurs, except that ejection reaches completion in vivo in Ͻ2 min (12). One study used lipid vesicles incorporating LamB and filled with ethidium bromide: the DNA was ejected into the vesicles, causing an increase in fluorescence over Ϸ30 s (13). However, the Ϸ1,000 molecules of ethidium bromide in each vesicle were enough for only the first 1 kbp of DNA (14). Also, because the ejections could have started at different times, that experiment says very little about the DNA translocation process. This paper aims to resolve these challenges in describing the ejection process.An important insight from theory is that frictional forces limit the speed of ejection, due to DNA rearrangement in the phage head or sliding forces in the tail (15,16). Because the DNA is in a liquid state (17), we expect friction to behave at least somewhat like macroscopic hydrodynamic drag: stronger at higher speed or at smaller spacings between the moving parts. The DNA-tail interaction does not change during the ejection process, so we expect friction in the tail to remain constant. In contrast, friction in the head should be stronger when the spacing between the loops of DNA is small, i.e., at the beginning of ejection.To quantify the rate of ejection, a single-phage technique is necessary. Single-phage ejections were first observed with fluorescence microscopy on phage T5, revealing an effect of the unique structure of the T5 genome: nicks in the DNA resulted in predefined stopping points and a stepwise translocation process, with speeds that were too high to be quantified, so that further analysis of the speed and source of friction was not possible (18). As we will show here, ejects...

We demonstrate the energy dependence of the motion of a porin, the lambda-receptor, in the outer membrane of living Escherichia coli by single molecule investigations. By poisoning the bacteria with arsenate and azide, the bacterial energy metabolism was stopped. The motility of individual lambda-receptors significantly and rapidly decreased upon energy depletion. We suggest two different causes for the ceased motility upon comprised energy metabolism: One possible cause is that the cell uses energy to actively wiggle its proteins, this energy being one order-of-magnitude larger than thermal energy. Another possible cause is an induced change in the connection between the lambda-receptor and the membrane structure, for instance by a stiffening of part of the membrane structure. Treatment of the cells with ampicillin, which directly targets the bacterial cell wall by inhibiting cross-linking of the peptidoglycan layer, had an effect similar to energy depletion and the motility of the lambda-receptor significantly decreased. Since the lambda-receptor is closely linked to the peptidoglycan layer, we propose that lambda-receptor motility is directly coupled to the constant and dynamic energy-consuming reconstruction of the peptidoglycan layer. The result of this motion could be to facilitate transport of maltose-dextrins through the porin.

Following the movement of individual molecules of a bacterial surface protein in vivo we investigated the effects of antibiotics and antimicrobial peptides on protein motility and membrane structure. In previous work we engineered the lambda-receptor of Escherichia coli such that less than one receptor per cell is in vivo biotinylated and can bind to a streptavidin coated bead. Such a bead served as a handle for the optical tweezers to follow the motion of an individual receptor. In an un-perturbed living cell the lambda-receptor performs a confined diffusive motion. The lambda-receptor links to the peptidoglycan layer, and indeed, a perturbation of the peptidoglycan layer had a pronounced effect on the motility of the receptor: The motility significantly decreases upon treatment with vancomycin or ampicillin, to study the effect of vancomycin we used strains with increased membrane permeability. As the motility of an individual receptor was monitored over an extended amount of time we were able to observe a temporal evolution of the action of vancomycin. Antimicrobial peptides (AMPs) are alternatives to conventional antibiotics in the treatment of bacterial infections. Therefore, we also investigated the effect of the toxic AMP polymyxin B (PMB) which targets both the outer and inner membranes and kills the organism. PMB significantly decreased the motility of the lambda-receptor. On the basis of these findings we confirm that the lambda-receptor is firmly attached to the peptidoglycan layer, and that an antibiotic or AMP mediated destruction of the dynamic peptidoglycan synthesis decreases the receptor motion.

Store-operated Ca 2þ entry (SOCE) is a mechanism that allows the entry of Ca 2þ upon depletion of the internal stores. The skeletal muscle cell is built for the rapid delivery of Ca 2þ to the contractile proteins. The cell microarchitecture allows this with the surface membrane invaginating into the cell forming the tubular (t-) system which apposes the sarcoplasmic reticulum (SR) for rapid signalling. In skeletal muscle SOCE has been shown to occur within 1 s of Ca 2þ release (Launikonis & Rios, 2007) but this should be significantly faster if the molecular agonists are prepositioned for activation. To examine SOCE kinetics we used skinned fibres from C57 mice (7-20 weeks old) with t-system trapped fluo-5N, bathed in an internal solution with 50 mM rhod-2. These dyes were simultaneously imaged in xyt mode on a confocal microscope (2 ms/line) while Ca 2þ release was induced by lowering [Mg 2þ ]. Global Ca 2þ release induced SOCE activation and deactivation as the Ca 2þ store refilled upon release inactivation (Launikonis & Rios, 2007). We also observed Ca 2þ waves in the continued presence of low Mg 2þ . These waves allowed an accurate observation of the latency between SR Ca 2þ release and SOCE. Thus SOCE ''coupling delay'' following the initiation of SR Ca 2þ release was determined to be 27 5 4 ms (n ¼ 6).