Chromosome replication does not trigger cell division in E. coli

An essential part of the chromosome replication origin of E. coli K-12 and B/r was replaced by the plasmid pOU71. The average initiation mass of replication for pOU71 decreases with increasing temperature. The constructed strains were grown exponentially at different temperatures, and cell sizes and DNA content were measured by flow cytometry. The average DNA content increased with increasing temperature, but the cell size distribution was largely unaffected. Furthermore, cells in which DNA replication had not yet initiated (cells in the B period) became less abundant with increasing temperature. The increased DNA content could not be explained by an increase in the length of the C period. It is concluded that chromosome replication does not trigger cell division in E. coli, but that the chromosome replication and cell division cycles of E. coli run in parallel independently of each other.     

N-Benzyl-3-sulfonamidopyrrolidines as novel inhibitors of cell division in E. coli

A new small molecule inhibitor of bacterial cell division has been discovered using a high-throughput screen in Escherichia coli. Although the lead screening hit (534F6) exhibited modest inhibition of the GTPase activity of FtsZ (20+/-5% at 100microM of compound), a primary target for bacterial cell division inhibitors, several analogs caused potent bacterial growth inhibition with negligible antagonism of FtsZ GTPase activity. A library of analogs has been prepared and several alkyne-tagged photoaffinity probes have been synthesized for use in experiments to elucidate the primary target of this compound.     

A MatP-divisome interaction coordinates chromosome segregation with cell division in E. coli

Initiation of chromosome segregation in bacteria is achieved by proteins acting near the origin of replication. Here, we report that the precise choreography of the terminus region of the Escherichia coli chromosome is also tightly controlled. The segregation of the terminus (Ter) macrodomain (MD) involves the structuring factor MatP. We characterized that migration of the Ter MD from the new pole to mid-cell and its subsequent persistent localization at mid-cell relies on several processes. First, the replication of the Ter DNA is concomitant with its recruitment from the new pole to mid-cell in a sequential order correlated with the position on the genetic map. Second, using a strain carrying a linear chromosome with the Ter MD split in two parts, we show that replisomes are repositioned at mid-cell when replication of the Ter occurs. Third, we demonstrate that anchoring the Ter MD at mid-cell depends on the specific interaction of MatP with the division apparatus-associated protein ZapB. Our results reveal how segregation of the Ter MD is integrated in the cell-cycle control.     

A multistranded polymer model explains MinDE dynamics in E. coli cell division

In Escherichia coli, the location of the site for cell division is regulated by the action of the Min proteins. These proteins undergo a periodic pole-to-pole oscillation that involves polymerization and ATPase activity of MinD under the controlling influence of MinE. This oscillation suppresses division near the poles while permitting division at midcell. Here, we propose a multistranded polymer model for MinD and MinE dynamics that quantitatively agrees with the experimentally observed dynamics in wild-type cells and in several well-studied mutant phenotypes. The model also provides new explanations for several phenotypes that have never been addressed by previous modeling attempts. In doing so, the model bridges a theoretical gap between protein structure, biochemistry, and mutant phenotypes. Finally, the model emphasizes the importance of nonequilibrium polymer dynamics in cell function by demonstrating how behavior analogous to the dynamic instability of microtubules is used by E. coli to achieve a sufficiently rapid timescale in controlling division site selection.     

Positioning of chemosensory clusters in E. coli and its relation to cell division

Chemotaxis receptors and associated signalling proteins in Escherichia coli form clusters that consist of thousands of molecules and are the largest native protein complexes described to date in bacteria. Clusters are located at the cell poles and laterally along the cell body, and play an important role in signal transduction. Much work has been done to study the structure and function of receptor clusters, but the significance of their positioning and the underlying mechanisms are not understood. Here, we used fluorescence imaging to study cluster distribution and follow cluster dynamics during cell growth. Our data show that lateral clusters localise to specific periodic positions along the cell body, which mark future division sites and are involved in the localisation of the replication machinery. The chemoreceptor cluster positioning is thus intricately related to the overall structure and division of an E. coli cell.     

Two pathways of division inhibition in UV-irradiated E. coli

We have investigated the mechanism of division inhibition in E. coli following UV-irradiation or nalidixic acid treatment. After UV, two separate mechanisms, both dependent upon recA+, appear to block division. One mechanism is dependent upon sfiA and sfiB, is inhibited by low levels (4 micrograms/ml) of rifamycin and is expressed in tif mutants at 42 degrees C. The second mechanism is independent of sfiA, and sfiB, is resistant to rifamycin and does not occur in cells lacking DNA replication forks. We suggest that this second mechanism is the result of the failure to terminate DNA replication in inhibited cells. Nalidixic acid inhibition of cell division also appears to involve both mechanisms but as found previously replication forks are also necessary to induce the sfi pathway.     

Regulation of adenylate cyclase in E. coli

The intracellular concentrations of cAMP in Escherichia coli are regulated mainly by control of the activity of adenylate cyclase. Withdrawal of the carbon source from the growth medium causes a gradual reduction of cellular energy and a dramatic stimulation of cyclase activity. Manipulations of the proton gradient at the cell membrane of ATP synthase-deficient E. coli (unc-) revealed that this part of the energy compartment is not responsible for the starvation-induced stimulation of cyclase. Neither is the ATP pool involved in regulation of the activity of the cyclase. The intracellular concentrations of ATP were experimentally lowered by purine starvation of auxotrophs, by inhibition of purine synthesis using amethopterin, or by affecting ATP synthesis using arsenate. None of these conditions led to stimulation of cyclase activity. The control of cyclase is exerted not via the energy pools but via uptake systems of energy substrates independent of whether the substrate can be metabolized or not, or how the transport is energized. The stringent coupling between these transport systems and cyclase activity enables the cell to react instantaneously to changes in its environment.     

TheEscherichia coli cell cycle, cell division and ppGpp: Regulation and mechanisms

The literature demonstrating tight regulation of theEscherichia coli cell cycle is reviewed. Recent evidence is presented indicating that the normal rod cell shape can be abandoned, allowing growth as a coccus, either by increasing the amount of the division proteins FtsZ, FtsA and FtsQ, or by increasing the pool of the nucleotide ppGpp. It is argued that ppGpp may be a cell cycle signal inE. coli.     

Regulation of cell division in Escherichia coli: properties of new ftsZ mutants

Summary Cells of Escherichia coli which produce high levels of the sfiA protein are UV-sensitive and filament extensively. It has been postulated that the sfiA protein is a division inhibitor which interacts with the ftsZ protein (formerly called sfiB or sulB) leading to cell division arrest. Under certain conditions, a similar division inhibition is observed with cells harboring a mutationally altered tsM allele, another division gene which was postulated to code for a division inhibitor or a controlling effector thereof (Drapeau et al.) (1984). In this communication, we report on the properties of ftsZ mutants isolated under conditions which brought no selective pressure. These mutants have either an increased sensitivity to UV irradiation or filament drastically following a nutritional shift-up, or both, or even cannot grow in a rich medium. They presumably possess a ftsZ protein which responds more readily to the inhibitory action of the wild type sfiA or the mutationally altered tsM1 protein since the phenotypic expressions associated with the mutations are not observed in the presence of the sfiA11 mutation or are amplified when the ftsZ mutant cells harbor the tsM1 allele. These results further support earlier suggestions that sfiA modulates ftsZ activity and establish tsM as an additional regulatory element thereof. In addition, it is shown that E. coli strain B is a naturally occurring ftsZ mutant.     

Two pathways of division inhibition in UV-irradiated E. coli

Molecular Genetics and Genomics - We have investigated the mechanism of division inhibition in E. coli following UV-irradiation or nalidixic acid treatment. After UV, two separate mechanisms, both...     

Process of cellular division in Escherichia coli growth pattern of E. coli murein

The growth pattern of the murein-sacculus which determines the shape of the Escherichia coli cell was studied by the use of high-resolution autoradiography with the electron microscope. The murein was pulse labelled with ³H-labelled diaminopimelic acid as a specific murein precursor and sacculi were prepared immediately. The radioactivity of the nascent murein appeared on the auto- radiographs at a well-defined growth zone in the central area of the sacculus. This was true regardless of the size of the cells. Pulse chase experimenta show rapid mixing of labelled murein with pre-existing murein and its even distribution over the whole surface of the sacculus.     

Cyclic AMP and cell division in Escherichia coli.

We examined several aspects of cell division regulation in Escherichia coli which have been thought to be controlled by cyclic AMP (cAMP) and its receptor protein (CAP). Mutants lacking adenyl cyclase (cya) or CAP (crp) were rod shaped, not spherical, during exponential growth in LB broth or glucose-Casamino Acids medium, and lateral wall elongation was normal; in broth, stationary-phase cells became ovoid. Cell mass was smaller for the mutants than for the wild type, but it remained appropriate for their slower growth rate and thus probably does not reflect early (uncontrolled) septation. The slow growth did not seem to reflect a gross metabolic disorder, since the mutants gave a normal yield on limiting glucose; surprisingly, however, the cya mutant (unlike crp) was unable to grow anaerobically on glucose, suggesting a role for cAMP (but not for CAP) in the expression of some fermentation enzyme. Both cya and crp mutants are known to be resistant to mecillinam, an antibiotic which inhibits penicillin-binding protein 2 (involved in lateral wall elongation) and also affects septation. This resistance does not reflect a lack of PBP2. Furthermore, it was not simply the result of slow growth and small cell mass, since small wild-type cells growing in acetate remained sensitive. The cAMP-CAP complex may regulate the synthesis of some link between PBP2 and the septation apparatus. The ftsZ gene, coding for a cell division protein, was expressed at a higher level in the absence of cAMP, as measured with an ftsZ::lacZ fusion, but the amount of protein per cell, shown by others to be invariable over a 10-fold range of cell mass, was independent of cAMP, suggesting that ftsZ expression is not regulated by the cAMP-CAP complex.     

大肠杆菌中外源基因的表达调节

大肠杆菌已经被广泛地应用于表达各种外源基因,但基因的表达受到多种因素的调节,而且不同的外源基因在大肠杆菌中的表达效率也有很大差异。本文从转录水平调节、翻译水平调节、培养条件调节等方面综述了大肠杆菌中外源基因的表达调节,以便认识其规律,有助于使用有效的方法提高外源基因在大肠杆菌中的表达效率。     

Low-temperature conditional cell division mutants of Escherichia coli.

Fifteen low-temperature conditional division mutants of Escherichia coli K-12 was isolated. They grew normally at 39 degrees C but formed filaments at 30 degrees C. All exhibited a coordinated burst of cell division when the filaments were shifted to the permissive temperature (39 degrees C). None of the various agents that stimulate cell division in other mutant systems (salt, sucrose, ethanol, and chloramphenicol) was very effective in restoring colony-forming ability at 25 degrees C or in stimulating cell division in broth. One of these mutants, strain JS10, was found to have an altered cell envelope as evidenced by increased sensitivity to deoxycholate and antibiotics, as well as leakage of ribonulcease I, a periplasmic enzyme. This mutant had normal rates of DNA synthesis, RNA synthesis, and phospholipid synthesis at both the nonpermissive and permissive temperatures. However, strain JS10 required new protein synthesis in the apparent absence of new RNA synthesis for division of filaments at the permissive temperature. The division of lesion in strain JS10 is cotransducible with malA, aroB, and glpD and maps within min 72 to 75 on the E. coli chromosome.