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      Choreography of the Mycobacterium Replication Machinery during the Cell Cycle

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          ABSTRACT

          It has recently been demonstrated that bacterial chromosomes are highly organized, with specific positioning of the replication initiation region. Moreover, the positioning of the replication machinery (replisome) has been shown to be variable and dependent on species-specific cell cycle features. Here, we analyzed replisome positions in Mycobacterium smegmatis, a slow-growing bacterium that exhibits characteristic asymmetric polar cell extension. Time-lapse fluorescence microscopy analyses revealed that the replisome is slightly off-center in mycobacterial cells, a feature that is likely correlated with the asymmetric growth of Mycobacterium cell poles. Estimates of the timing of chromosome replication in relation to the cell cycle, as well as cell division and chromosome segregation events, revealed that chromosomal origin-of-replication ( oriC) regions segregate soon after the start of replication. Moreover, our data demonstrate that organization of the chromosome by ParB determines the replisome choreography.

          IMPORTANCE

          Despite significant progress in elucidating the basic processes of bacterial chromosome replication and segregation, understanding of chromosome dynamics during the mycobacterial cell cycle remains incomplete. Here, we provide in vivo experimental evidence that replisomes in Mycobacterium smegmatis are highly dynamic, frequently splitting into two distinct replication forks. However, unlike in Escherichia coli, the forks do not segregate toward opposite cell poles but remain in relatively close proximity. In addition, we show that replication cycles do not overlap. Finally, our data suggest that ParB participates in the positioning of newly born replisomes in M. smegmatis cells. The present results broaden our understanding of chromosome segregation in slow-growing bacteria. In view of the complexity of the mycobacterial cell cycle, especially for pathogenic representatives of the genus, understanding the mechanisms and factors that affect chromosome dynamics will facilitate the identification of novel antimicrobial factors.

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          Most cited references47

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          High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics.

          Bacteria display various shapes and rely on complex spatial organization of their intracellular components for many cellular processes. This organization changes in response to internal and external cues. Quantitative, unbiased study of these spatio-temporal dynamics requires automated image analysis of large microscopy datasets. We have therefore developed MicrobeTracker, a versatile and high-throughput image analysis program that outlines and segments cells with subpixel precision, even in crowded images and mini-colonies, enabling cell lineage tracking. MicrobeTracker comes with an integrated accessory tool, SpotFinder, which precisely tracks foci of fluorescently labelled molecules inside cells. Using MicrobeTracker, we discover that the dynamics of the extensively studied Escherichia coli Min oscillator depends on Min protein concentration, unveiling critical limitations in robustness within the oscillator. We also find that the fraction of MinD proteins oscillating increases with cell length, indicating that the oscillator has evolved to be most effective when cells attain an appropriate length. MicrobeTracker was also used to uncover novel aspects of morphogenesis and cell cycle regulation in Caulobacter crescentus. By tracking filamentous cells, we show that the chromosomal origin at the old-pole is responsible for most replication/separation events while the others remain largely silent despite contiguous cytoplasm. This surprising position-dependent silencing is regulated by division. © 2011 Blackwell Publishing Ltd.
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            Asymmetry and aging of mycobacterial cells lead to variable growth and antibiotic susceptibility.

            Cells use both deterministic and stochastic mechanisms to generate cell-to-cell heterogeneity, which enables the population to better withstand environmental stress. Here we show that, within a clonal population of mycobacteria, there is deterministic heterogeneity in elongation rate that arises because mycobacteria grow in an unusual, unipolar fashion. Division of the asymmetrically growing mother cell gives rise to daughter cells that differ in elongation rate and size. Because the mycobacterial cell division cycle is governed by time, not cell size, rapidly elongating cells do not divide more frequently than slowly elongating cells. The physiologically distinct subpopulations of cells that arise through asymmetric growth and division are differentially susceptible to clinically important classes of antibiotics.
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              Stoichiometry and architecture of active DNA replication machinery in Escherichia coli.

              The multiprotein replisome complex that replicates DNA has been extensively characterized in vitro, but its composition and architecture in vivo is unknown. Using millisecond single-molecule fluorescence microscopy in living cells expressing fluorescent derivatives of replisome components, we have examined replisome stoichiometry and architecture. Active Escherichia coli replisomes contain three molecules of the replicative polymerase, rather than the historically accepted two. These are associated with three molecules of tau, a clamp loader component that trimerizes polymerase. Only two of the three sliding clamps are always associated with the core replisome. Single-strand binding protein has a broader spatial distribution than the core components, with 5 to 11 tetramers per replisome. This in vivo technique could provide single-molecule insight into other molecular machines.
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                Author and article information

                Journal
                mBio
                MBio
                mbio
                mbio
                mBio
                mBio
                American Society of Microbiology (1752 N St., N.W., Washington, DC )
                2150-7511
                17 February 2015
                Jan-Feb 2015
                : 6
                : 1
                : e02125-14
                Affiliations
                [a ]Department of Molecular Microbiology, Faculty of Biotechnology, University of Wrocław, Wrocław, Poland
                [b ]Department of Biochemistry, Microbiology Unit, University of Oxford, Oxford, United Kingdom
                [c ]Department of Microbiology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland
                Author notes
                Address correspondence to Jolanta Zakrzewska-Czerwińska, jolanta.zakrzewska@ 123456uni.wroc.pl .

                K.G. and M.P. contributed equally to this work.

                Invited Editor William Margolin, University of Texas Medical School at Houston Editor Steven J. Norris, University of Texas Medical School at Houston

                Article
                mBio02125-14
                10.1128/mBio.02125-14
                4337567
                25691599
                a58cc041-8770-4ea7-a65d-0a751bbfded7
                Copyright © 2015 Trojanowski et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 10 October 2014
                : 23 December 2014
                Page count
                supplementary-material: 0, Figures: 7, Tables: 0, Equations: 0, References: 50, Pages: 12, Words: 9118
                Categories
                Research Article
                Custom metadata
                January/February 2015

                Life sciences
                Life sciences

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