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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: To distinguish between a gradual or an abrupt movement of the Escherichia coli nucleoid during partitioning we determined the distances between nucleoid borders and cell poles. Measurements were performed on fixed but hydrated cells and on living cells growing in steady state. The distance between nucleoid outer border and cell pole remained constant in cells with either one or two nucleoids. Thus the nucleoid outer borders moved gradually during the partition process. To study partitioning during recovery from protein-synthesis inhibition cells were treated with chloramphenicol. After growth resumption, cells and nucleoids first elongated before partitioning occurred. Again, no indication of a rapid displacement of the nucleoid to one-quarter and three-quarter positions in the cell was observed.
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  • 2
    ISSN: 1432-072X
    Keywords: Escherichia coli ; Temperature ; Morphology ; Cell shape ; Cell size
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two substrains of Escherichia coli B/r were grown to steady-state in batch cultures at temperatures between 22 and 42° C in different growth media. The size and shape of the cells were measured from light and electron micrographs and with the Coulter channelizer. The results indicate that cells are shorter and somewhat thicker at the lower temperatures, especially in rich growth media; cell volume is then slightly smaller. A lower temperature was further found to increase the relative duration of the constriction period. The shapes of the cell size distributions are indistinguishable, indicating that the pattern of growth of the cells is the same at all temperatures. The adaptation of the cells to a temperature shift lasted several generations, indicating that the morphological effects of temperature are mediated by the cell's physiology.
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  • 3
    ISSN: 1617-4623
    Keywords: Escherichia coli ; minB ; Minicells ; Segregation ; Supercoiling
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Nucleoid segregation in the Escherichia coli minB mutant and in cells that over-produce minB gene products appeared defective as measured from fluorescence micrographs. Electrophoretic resolution of topoisomers of plasmid isolates from the minB strain revealed a decreased level of negative supercoiling; in addition, multimerization was observed. Over-production of the minB gene product also resulted in a decreased level of negative supercoiling. This phenotype is typical of the gyrB(ts) mutant, which is known to be affected in chromosome decatenation and supercoiling. We propose that the minB mutation and over-production of the minB gene products cause a defect in nucleoid segregation, which may be related to the decrease in negative supercoiling. As in the gyrB(ts) mutant, retardation of nucleoid segregation is proposed to inhibit constriction initiation in the cell centre and to give rise to nucleoid-free cell poles. As a consequence, these cells divide between nucleoid and cell pole, resulting in minicell and (sometimes) in anucleate cell formation.
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  • 4
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The Escherichia coli DNA replication genes dnaZ and dnaX have previously been localized very near each other at 10.4 to 10.5 min on the chromosome map. These genes were cloned from a dnaZ + X + plasmid of the Clarke and Carbon collection by identifying complementing fragments and both were located on a 2.1 kilobase pair (kb) fragment. The organization of the Z and X genes was investigated by Tn5 mutagenesis of a Z + Y + plasmid. Insertions which abolished Z or X complementing activity were mapped by restriction enzyme analysis within the 2.1 kb fragment. With the exception of one atypical insertion, all the insertions inactivated both Z and X complementation. The protein products of the dnaZ-dnaX region were labelled in minicells containing dnaZ + X + and dnaZX:: Tn5 plasmids. The 2.1 kb ZX region (which has a maximum coding capacity of 77,000 daltons of protein in a single reading frame) directed the synthesis of two proteins, one of 75,000 daltons, designated dnaX, and another of 56,500 daltons, designated dnaZ. Tn5 insertion into the ZX region interrupted the synthesis of these proteins; the detection of truncated fragments of dnaX determined the direction of transcription. In vitro, using a coupled transcription-translation system dependent on plasmid DNA, synthesis of the 75,000 dalton dnaX protein was demonstrated, but there was no detectable synthesis of the smaller dnaZ protein. Probably, therefore, the 75,000 dalton dnaX protein is cleaved in vivo to generate the dnaZ protein. It is possible that the 75,000 dalton product is the τ subunit of DNA polymerase III because they migrated similarly in electrophoresis.
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 226 (2003), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The spatial relationship between FtsZ localization and nucleoid segregation was followed in Escherichia coli thyA cells, made spheroidal by brief exposure to mecillinam and after manipulating chromosome replication time using changes (‘steps’) in thymine concentration [Zaritsky et al., Microbiology 145 (1999) 1015–1022]. In such cells, fluorescent FtsZ-GFP arcs did not overlap the DAPI-stained nucleoids. It is concluded that FtsZ rings are deposited between segregating nucleoids, consistent with the nucleoid occlusion model [Woldringh et al., J. Bacteriol. 176 (1994) 6030–6038].
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  • 6
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract The mechanisms that determine chromosome structure and chromosome partitioning in bacteria are largely unknown. Here we discuss two hypotheses: (i) the structure of the Escherichia coli nucleoid is determined by DNA binding proteins and DNA supercoiling, representing a compaction force on the one hand, and by the coupled transcription/translation/ translocation of plasma membrane and cell wall proteins, representing an expansion force on the other hand; (ii) the two forces are important for the partitioning process of chromosomes.
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  • 7
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Many recent reviews in the field of bacterial chromosome segregation propose that newly replicated DNA is actively separated by the functioning of specific proteins. This view is primarily based on an interpretation of the position of fluorescently labelled DNA regions and proteins in analogy to the active segregation mechanism in eukaryotic cells, i.e. to mitosis. So far, physical aspects of DNA organization such as the diffusional movement of DNA supercoil segments and their interaction with soluble proteins, leading to a phase separation between cytoplasm and nucleoid, have received relatively little attention. Here, a quite different view is described taking into account DNA–protein interactions, the large variation in the cellular position of fluorescent foci and the compaction and fusion of segregated nucleoids upon inhibition of RNA or protein synthesis. It is proposed that the random diffusion of DNA supercoil segments is transiently constrained by the process of co- transcriptional translation and translocation (transertion) of membrane proteins. After initiation of DNA replication, a bias in the positioning of transertion areas creates a bidirectionality in chromosome segre-gation that becomes self-enhanced when neigh-bouring genes on the same daughter chromosome are expressed. This transertion-mediated segregation model is applicable to multifork replication during rapid growth and to multiple chromosomes and plasmids that occur in many bacteria.
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  • 8
    Electronic Resource
    Electronic Resource
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 32 (1999), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Bacterial 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 4′,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.
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 14 (1994), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: During hyperosmotic shock, the protoplast and stretched-out peptidoglycan layer first shrink together until the turgor pressure in the cell is relieved. Being non-compressible, the outer and inner membranes must fold their superfluous surfaces. While the protoplast contracts further, the inner membrane rearranges into plasmolysis spaces visible by phase contrast microscopy. Two opposing theories predict a similar positioning of spaces in dividing cells and filaments: the ‘periseptal annulus model, based on adhesion zones, involved in the predetermination of the division site; and a ‘restricted, random model’, based on physical properties of the protoplast. Strong osmotic shock causes retraction of the inner membrane over almost the entire surface forming the so-called ‘Bayer bridges’. These tubular adhesion sites are preserved by chemical fixation, and can be destroyed by cryofixation and freeze-substitution of unfixed ceils. Both the regular positioning of the plasmolysis spaces and the occurrence of tubular adhesion sites can be explained on the basis of physical properties of the membrane which necessitate rearrangements by membrane flow during shrinkage of the protoplast.
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  • 10
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; vacuole biogenesis ; phenotypic lag ; optical trapping ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The conditional vacuolar segregation mutant vac2-1 [Shaw and Wickner (1991) EMBO J. 10,1741-1748] shifted to non-permissive temperature (37°C), forms large-budded cells without a vacuole in the bud, and daughter cells without an apparent vacuole. Some cells still contain normal segregation structures. Structural and biochemical quantification of the segregation defect showed that (i) about 10% of the full-grown buds did not contain a vacuole, (ii) about 15% of the small cells washed out of a population growing in an elutriation chamber at 37°C, did not contain a visible vacuole, and (iii) 15% of the cells per generation lost carboxypeptidase Y activity after proteinase A depletion. Thus, 10-15% of the daughter cells did not inherit vacuolar structures or vacuolar proteolytic activity from the mother cell. To investigate the fate of vacuole-less daughters, these cells were isolated by optical trapping. The isolated cells formed colonies on agar plates that consisted of cells with normal vacuoles, both at 23 and 37°C. Thus, the vacuole-less cells that failed to inherit proteolytic activities from the mother cell apparently give rise to progeny containing structurally normal vacuoles. Time-lapse experiments showed that vacuole-less daughter cells formed vacuolar vesicles that fused into a new vacuole within 30 min. Although new buds only emerged after a vacuole had formed in the mother cell, the temporary lack of a vacuole had little effect on growth rate. The results suggest that an alternative pathway for vacuole formation exists, and that yeast cells may require a vacuole of some minimal size to initiate a new round of budding. © 1997 by John Wiley & Sons, Ltd.
    Additional Material: 4 Ill.
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