Several parameters of phage T4 adsorption to and growth in Escherichia coli B/r were determined. All changed monotonously with the bacterial growth rate (p), which was modified by nutritional conditions. Adsorption rate was faster at higher p values, positively correlated to cell size, and increased by pretreatment with low penicillin (Pn) concentrations; it was directly proportional to total cellular surface area, indicating a constant density of 14 receptors on cell envelopes irrespective of growth conditions. Parameters of phage development and cell lysis were pdependent. The rate of phage release and burst size increased, while the eclipse and latent periods decreased with increasing p. Differentiation between the contribution of several physiological parameters to the development of 14 was performed by manipulating the host cells. A competitive inhibitor of glucose uptake, methyl a-o-glucoside, was exploited to reduce the growth rate in the same effective carbon source. Synchronous cells were obtained by the 'baby-machine' and large cells were obtained by pretreatment with low Pn concentrations. Lysis was delayed by superinfection, and DNA content and concentration were modified by growing a thy mutant in limiting thymine concentrations. The results indicate that burst size is not limited by cell size or DNA composition, nor directly by the rate of metabolism, but rather by the rates of synthesis and assembly of phage components and by lysis time. The rates of synthesis and assembly of phage components seem to depend on the content of the protein-synthesizing system and lysis time seems to depend on cellular dimensions.

The peripheral membrane ATPase MinD is a component of the Min system responsible for correct placement of the division site in Escherichia coli cells. By rapidly migrating from one cell pole to the other, MinD helps to block unwanted septation events at the poles. MinD is an amphitropic protein that is localized to the membrane in its ATP-bound form. A C-terminal domain essential for membrane localization is predicted to be an amphipathic ␣-helix with hydrophobic residues interacting with lipid acyl chains and cationic residues on the opposite face of the helix interacting with the head groups of anionic phospholipids (Szeto, T. H., Rowland, S. L., Rothfield, L. I., and King, G. F.

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.

SummaryBacterial membrane and nucleoids were stained concurrently by the lipophilic styryl dye FM 4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl) hexatrienyl)pyridinium dibromide] and 4Ј,6-diamidino-2-phenylindole (DAPI), respectively, and studied using fluorescence microscopy imaging. Observation of plasmolysed cells indicated that FM 4-64 stained the inner membrane preferentially. In live Escherichia coli pbpB cells and filaments, prepared on wet agar slabs, an FM 4-64 staining pattern developed in the form of dark bands. In dividing cells, the bands occurred mainly at the constriction sites and, in filaments, between partitioning nucleoids. The FM 4-64 pattern of dark bands in filaments was abolished after inhibiting protein synthesis with chloramphenicol. It is proposed that the staining patterns reflect putative membrane domains formed by DNA-membrane interactions and have functional implications in cell division.

It is crucial to the reproducibility of results and their proper interpretation that the conditions under which experiments are carried out be defined with rigour and consistency. In this review we attempt to clarify the differences and interrelationships among steady, balanced and exponential states of culture growth. Basic thermodynamic concepts are used to introduce the idea of steady-state growth in open, biological systems. The classical, sometimes conflicting, definitions of steady-state and balanced growth are presented, and a consistent terminology is proposed. The conditions under which a culture in balanced growth is also in exponential growth and in steady-state growth are indicated. It is pointed out that steady-state growth always implies both balanced and exponential growth, and examples in which the converse does not hold are described. More complex situations are then characterized and the terminology extended accordingly. This leads to the notion of normal growth and growth that can be synchronous or otherwise unbalanced but still reproducible, and to the condition of approximate steady state manifested by growth in batch culture and by asymmetrically dividing cells, which is analysed in some detail.

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.

SummaryCell cycle events have been proposed to be triggered by the formation of membrane domains in the process of coupled transcription, translation and insertion ('transertion') of nascent membrane and exported proteins. Disruption of domain structure should lead to changes in membrane dynamics. Membrane viscosity of Escherichia coli and Bacillus subtilis decreased after inhibition of protein synthesis by chloramphenicol or puromycin, or of RNA initiation by rifampicin, but not after inhibition of RNA elongation by streptolydigin or amino acid starvation of a stringent strain. The decrease caused by inhibitors of protein synthesis was prevented by streptolydigin if added simultaneously, but was not reversed if added later. The drug-induced decrease in membrane viscosity is energy dependent: it did not happen in KCN-treated cells. All treatments decreasing membrane viscosity also induced nucleoid compaction and fusion. Inhibition of macromolecular synthesis without membrane perturbation caused nucleoids to expand. Changes in membrane dynamics were also displayed during a nutritional shift-down transition that causes imbalance in macromolecular syntheses. The results are consistent with the transertion model, predicting dissipation of membrane domains by termination of protein synthesis or detachment of polysomes from DNA; domain structure is conserved if the transertion process is 'frozen'.

DnaA is the initiator protein for chromosomal replication in bacteria; its activity plays a central role in the timing of the primary initiations within the Escherichia coli cell cycle. A controlled, reversible conversion between the active ATP-DnaA and the inactive ADP forms modulates this activity. In a DNA-dependent manner, bound ATP is hydrolyzed to ADP. Acidic phospholipids with unsaturated fatty acids are capable of reactivating ADP-DnaA by promoting the release of the tightly bound ADP. The nucleotide dissociation kinetics, measured in the present study with the fluorescent derivative 3-O-(N-methylantraniloyl)-5-adenosine triphosphate, was dependent on the density of DnaA on the membrane in a cooperative manner: it increased 5-fold with decreased protein density. At all surface densities the nucleotide was completely released, presumably due to protein exchange on the membrane. Distinct temperature dependences and the effect of the crowding agent Ficoll suggest that two functional states of DnaA exist at high and low membrane occupancy, ascribed to local macromolecular crowding on the membrane surface. These novel phenomena are thought to play a major role in the mechanism regulating the initiation of chromosomal replication in bacteria.