N. Nanninga

Cell division and peptidoglycan assembly in Escherichia coli.

Research on bacterial cell division has recently gained renewed impetus because of new information about peptidoglycan assembly and about specific cell‐division genes and their products. This paper concerns aspects of cell division that specifically concern the peptidoglycan. It is shown that upon division, peptidoglycan assembly switches from lateral wall location to the cell centre, that assembly takes place at the leading edge of the invaginating constriction, that the mode of glycan strand insertion changes from a single‐stranded mode to a multi‐stranded mode, and that the initiation of division (in contrast to its continuation) requires penicillin‐insensitive peptidoglycan synthesis (PIPS). A membrane component X (possibly FtsQ) is proposed to coordinate PIPS with the cell division‐initiating protein FtsZ. It is suggested that a largely proteinaceous macromolecular complex (divisome) at the leading edge of constriction encompasses three compartments (cytoplasm, membrane and periplasm). The composition of this complex is proposed to vary depending on whether division is being initiated or completed.

Penicillin-binding protein PBP2 of Escherichia coli localizes preferentially in the lateral wall and at mid-cell in comparison with the old cell pole.

The localization of penicillin‐binding protein 2 (PBP2) in Escherichia coli has been studied using a functional green fluorescent protein (GFP)–PBP2 fusion protein. PBP2 localized in the bacterial envelope in a spot‐like pattern and also at mid‐cell during cell division. PBP2 disappeared from mid‐cell just before separation of the two daughter cells. It localized with a preference for the cylindrical part of the bacterium in comparison with the old cell poles, which are known to be inert with respect to peptidoglycan synthesis. In contrast to subunits of the divisome, PBP2 failed to localize at mid‐cell when PBP3 was inhibited by the specific antibiotic aztreonam. Therefore, despite its dependency on active PBP3 for localization at mid‐cell, it seems not to be an integral part of the divisome. Cells grown for approximately half a mass doubling time in the presence of the PBP2 inhibitor mecillinam synthesized nascent cell poles with an increased diameter, indicating that PBP2 is required for the maintenance of the correct diameter of the new cell pole.

Escherichia coli Minicell Membranes Are Enriched in Cardiolipin

The phospholipid composition of Escherichia coli minicells has been studied as a model for the cell division site. Minicells appeared to be enriched in cardiolipin at the expense of phosphatidylglycerol. Mass spectrometry showed no differences between the gross acyl chain compositions of minicells and wild-type cells.

Topography of peptidoglycan synthesis during elongation and polar cap formation in a cell division mutant of Escherichia coli MC4100

SUMMARY: A cell division mutant of Escherichia coli K12 lysA, the temperature sensitive ftsZ strain, was pulse-labelled with [3H]diaminopimelic acid (DAP) during growth in minimal salts medium both at the permissive (28°C) and restrictive (42°C) temperature. In contrast to other known cell division mutants, ftsZ filaments obtained during growth at 42°C show no sign of persisting or newly initiated constrictions. The location of the incorporated DAP in dividing cells and in filaments was analysed with an improved autoradiographic method in which preparations of well-spread sacculi are covered with a dry emulsion. From the populations of sacculi complete distributions were obtained, which compared well with those of the intact cells. The grain-density distributions of cells dividing at 28°C showed that the rate of surface synthesis was strongly increased at the site of constriction at the expense of the activity in the lateral wall, suggesting a redistribution of surface synthesis activity. In individual filaments elongating at 42°C no indication for the existence of narrow or broad growth zones was found, suggesting a dispersed mode of lateral wall synthesis. These observations are in accordance with theoretical predictions on the rate of surface synthesis during the constriction period in cells which elongate at a constant diameter.

Escherichia coli contains a DNA replication compartment in the cell center.

The active replication forks of E. coli B/r K cells growing with a doubling time of 210 min have been pulse-labeled with [(3)H] thymidine for 10 min. By electron-microscopic autoradiography the silver grains have been localized in the various length classes. From the known pattern of the DNA replication period in the cell cycle at slow growth and from the average position of grains per length class it was deduced that DNA replication starts in the cell center and that it remains there for a substantial part of the DNA replication period. This suggests the occurrence of a centrally located DNA replication compartment.

Three‐dimensional imaging in fluorescence by confocal scanning microscopy

The improved resolution and sectioning capability of a confocal microscope make it an ideal instrument for extracting three‐dimensional information especially from extended biological specimens. The imaging properties, also with finite detection pinholes are considered and a number of biological applications demonstrated.

Three-dimensional confocal fluorescence microscopy.

Publisher Summary Biological material is organized in four dimensions: three spatial ones and a temporal one. Light microscopy is able to visualize biological objects in their natural watery condition and during their temporal development. Improved imaging is the optical sectioning property by which the contributions from out-of-focus areas in the specimen are effectively suppressed. In normal microscopy, these contributions lead to a strong reduction in the available image contrast. A three-dimensional microscope is obtained where each data point as collected represents the quantity of the specific contrast parameter used at a certain point in space. Deconvolution techniques have been developed for eliminating the out-of-focus information from conventional fluorescence microscopy. Confocal microscopy can deliver directly clear optical sections without the use of time-consuming image reconstruction algorithms. Image processing can also be used to enhance the confocal images. Computer-generated stereoscopic images are also used for the visualization of the three-dimensional biological information. This chapter discusses the number of aspects of confocal imaging, especially the advantages and drawbacks of the various scanning approaches.

DNA and origin region segregation are not affected by the transition from rod to sphere after inhibition of Escherichia coli MreB by A22.

The bacterial actin homologue MreB forms a helix underneath the cytoplasmic membrane and was shown to be essential in the morphogenesis of the rod‐shaped cells. Additionally, MreB was implicated to be involved in DNA segregation. However, in our hands the mreBCD deletion strain (PA340‐678) grew without apparent DNA segregation defect, suggesting that the reported chromosome segregation inhibition could be caused by a temporarily effect of MreB inhibition or depletion. To assess the involvement of MreB in DNA segregation during the transition from rod to sphere, we compared the effect of A22 and the PBP2 inhibitor mecillinam on the percentage of cells with segregated nucleoids and the number of oriC foci in wild‐type Escherichia coli cells. Cells became spherical in the same time window during both treatments and we could not detect any difference in the chromosome or oriC segregation between these two treatments. Additionally, flow cytometric analyses showed that A22 and mecillinam treatment gave essentially the same chromosome segregation pattern. We conclude that MreB is not directly involved in DNA segregation of E. coli.

R174 of Escherichia coli FtsZ is involved in membrane interaction and protofilament bundling, and is essential for cell division

We investigated the interaction between FtsZ and the cytoplasmic membrane using inside‐out vesicles. Comparison of the trypsin accessibility of purified FtsZ and cytoplasmic membrane‐bound FtsZ revealed that the protruding loop between helix 6 and helix 7 is protected from trypsin digestion in the latter. This hydrophobic loop contains an arginine residue at position 174. To investigate the role of R174, this residue was replaced by an aspartic acid, and FtsZ‐R174D was fused to green fluorescent protein (GFP). FtsZ‐R174D‐GFP could localize in an FtsZ and in an FtsZ84(Ts) background at both the permissive and the non‐permissive temperature, and it had a reduced affinity for the cytoplasmic membrane compared with wild‐type FtsZ. FtsZ‐R174D could also localize in an FtsZ depletion strain. However, in contrast to wild‐type FtsZ, FtsZ‐R174D was not able to complement the ftsZ84 mutation or the depletion strain and induced filamentation. In vitro polymerization experiments showed that FtsZ‐R174D is able to polymerize, but that these polymers cannot form bundles in the presence of 10 mM CaCl2. This is the first description of an FtsZ mutant that has reduced affinity for the cytoplasmic membrane and does not support cell division, but is still able to localize. The mutant is able to form protofilaments in vitro but fails to bundle. It suggests that neither membrane interaction nor bundling is a requirement for initiation of cell division.

Localization of T-DNA Insertions in Petunia by Fluorescence in Situ Hybridization: Physical Evidence for Suppression of Recombination.

Using fluorescence in situ hybridization (FISH) with metaphase preparations, we localized a 4-kb single-copy T-DNA sequence in a group of petunia transformants. The selected T-DNAs previously had been shown to be linked to the phenotypic marker FI on chromosome II. Linkage analysis had revealed that recombination around the FI locus is suppressed in a wide cross relative to an inbred recombination assay. The localization of six FI-linked T-DNAs and the FI locus itself, using FISH, revealed a number of aspects of recombination in petunia: (1) the central region of chromosome II showed at least a 10-fold suppression of recombination in wide crosses relative to the distal region; (2) recombination in wide hybrids over two-thirds of the chromosome was extremely low; and (3) recombination between completely homologous chromosomes in an inbred cross also was suppressed in the central region. In addition, the T-DNAs were not evenly distributed along the chromosome, suggesting a possible preference for a distal position for T-DNA integration. Implications for such a preference are discussed.