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1.  A Replisome’s journey through the bacterial chromosome 
Genome duplication requires the coordinated activity of a multi-component machine, the replisome. In contrast to the background of metabolic diversity across the bacterial domain, the composition and architecture of the bacterial replisome seem to have suffered few changes during evolution. This immutability underlines the replisome’s efficiency in copying the genome. It also highlights the success of various strategies inherent to the replisome for responding to stress and avoiding problems during critical stages of DNA synthesis. Here we summarize current understanding of bacterial replisome architecture and highlight the known variations in different bacterial taxa. We then look at the mechanisms in place to ensure that the bacterial replisome is assembled appropriately on DNA, kept together during elongation, and disassembled upon termination. We put forward the idea that the architecture of the replisome may be more flexible that previously thought and speculate on elements of the replisome that maintain its stability to ensure a safe journey from origin to terminus.
PMCID: PMC4456610  PMID: 26097470
DNA replication; replisome; bacteria; chromosome; evolution; DNA polymerase; Escherichia coli; Bacillus subtilis
2.  Slow unloading leads to DNA-bound β2-sliding clamp accumulation in live Escherichia coli cells 
Nature Communications  2014;5:5820.
The ubiquitous sliding clamp facilitates processivity of the replicative polymerase and acts as a platform to recruit proteins involved in replication, recombination and repair. While the dynamics of the E. coli β2-sliding clamp have been characterized in vitro, its in vivo stoichiometry and dynamics remain unclear. To probe both β2-clamp dynamics and stoichiometry in live E. coli cells, we use custom-built microfluidics in combination with single-molecule fluorescence microscopy and photoactivated fluorescence microscopy. We quantify the recruitment, binding and turnover of β2-sliding clamps on DNA during replication. These quantitative in vivo results demonstrate that numerous β2-clamps in E. coli remain on the DNA behind the replication fork for a protracted period of time, allowing them to form a docking platform for other enzymes involved in DNA metabolism.
DNA replication is accomplished by the replisome, a multi-protein complex that comprises the sliding clamp. Here, Moolman et al. present quantitative and dynamic measurements of the number of β 2-sliding clamps at the single-cell level in live E. coli cells to shed light on key aspects of DNA replication.
PMCID: PMC4284645  PMID: 25520215
3.  In vivo Architecture and Action of Bacterial Structural Maintenance of Chromosome Proteins 
Science (New York, N.Y.)  2012;338(6106):10.1126/science.1227126.
SMC (Structural Maintenance of Chromosome) proteins act ubiquitously in chromosome processing. In Escherichia coli, the SMC complex, MukBEF, plays roles in chromosome segregation and organization. We used single-molecule millisecond multicolor fluorescence microscopy of live bacteria to reveal that a dimer of dimeric fluorescent MukBEF molecules act as the minimal functional unit. 8-10 of these complexes, on average, accumulated as ‘spots’ in 1-3 discrete chromosome-associated regions of the cell, where they formed higher-order structures. Functional MukBEF within spots exchanged with freely diffusing complexes at a rate of one complex every ~50 s in reactions requiring ATP hydrolysis. Thus, by functioning in pairs, MukBEF complexes may undergo multiple cycles of ATP-hydrolysis without being released from DNA, analogous to the behavior of well-characterized molecular motors.
PMCID: PMC3807729  PMID: 23112333
4.  High-copy bacterial plasmids diffuse in the nucleoid-free space, replicate stochastically and are randomly partitioned at cell division 
Nucleic Acids Research  2013;42(2):1042-1051.
Bacterial plasmids play important roles in the metabolism, pathogenesis and bacterial evolution and are highly versatile biotechnological tools. Stable inheritance of plasmids depends on their autonomous replication and efficient partition to daughter cells at cell division. Active partition systems have not been identified for high-copy number plasmids, and it has been generally believed that they are partitioned randomly at cell division. Nevertheless, direct evidence for the cellular location of replicating and nonreplicating plasmids, and the partition mechanism has been lacking. We used as model pJHCMW1, a plasmid isolated from Klebsiella pneumoniae that includes two β-lactamase and two aminoglycoside resistance genes. Here we report that individual ColE1-type plasmid molecules are mobile and tend to be excluded from the nucleoid, mainly localizing at the cell poles but occasionally moving between poles along the long axis of the cell. As a consequence, at the moment of cell division, most plasmid molecules are located at the poles, resulting in efficient random partition to the daughter cells. Complete replication of individual molecules occurred stochastically and independently in the nucleoid-free space throughout the cell cycle, with a constant probability of initiation per plasmid.
PMCID: PMC3902917  PMID: 24137005
5.  Small Plasmids Harboring qnrB19: a Model for Plasmid Evolution Mediated by Site-Specific Recombination at oriT and Xer Sites 
Plasmids pPAB19-1, pPAB19-2, pPAB19-3, and pPAB19-4, isolated from Salmonella and Escherichia coli clinical strains from hospitals in Argentina, were completely sequenced. These plasmids include the qnrB19 gene and are 2,699, 3,082, 2,989, and 2,702 nucleotides long, respectively, and they share extensive homology among themselves and with other previously described small qnrB19-harboring plasmids. The genetic environment of qnrB19 in all four plasmids is identical to that in these other plasmids and in transposons such as Tn2012, Tn5387, and Tn5387-like. Nucleotide sequence comparisons among these and previously described plasmids showed a variable region characterized by being flanked by an oriT locus and a Xer recombination site. We propose that this arrangement could play a role in the evolution of plasmids and present a model for DNA swapping between plasmid molecules mediated by site-specific recombination events at oriT and a Xer target site.
PMCID: PMC3318318  PMID: 22290975
6.  The Escherichia coli SMC Complex, MukBEF, Shapes Nucleoid Organization Independently of DNA Replication 
Journal of Bacteriology  2012;194(17):4669-4676.
SMC (structural maintenance of chromosomes) complexes function ubiquitously in organizing and maintaining chromosomes. Functional fluorescent derivatives of the Escherichia coli SMC complex, MukBEF, form foci that associate with the replication origin region (ori). MukBEF impairment results in mispositioning of ori and other loci in steady-state cells. These observations led to an earlier proposal that MukBEF positions new replicated sister oris. We show here that MukBEF generates and maintains the cellular positioning of chromosome loci independently of DNA replication. Rapid impairment of MukBEF function by depleting a Muk component in the absence of DNA replication leads to loss of MukBEF foci as well as mispositioning of ori and other loci, while rapid Muk synthesis leads to rapid MukBEF focus formation but slow restoration of normal chromosomal locus positioning.
PMCID: PMC3415497  PMID: 22753058
7.  Stoichiometry and architecture of active DNA replication machinery in Escherichia coli * 
Science (New York, N.Y.)  2010;328(5977):498-501.
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 YPet 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 τ, 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 five to eleven tetramers per replisome. This in vivo technique could provide single molecule insight into other molecular machines.
PMCID: PMC2859602  PMID: 20413500
8.  Independent Positioning and Action of Escherichia coli Replisomes in Live Cells 
Cell  2008;133(1):90-102.
A prevalent view of DNA replication has been that it is carried out in fixed “replication factories.” By tracking the progression of sister replication forks with respect to genetic loci in live Escherichia coli, we show that at initiation replisomes assemble at replication origins irrespective of where the origins are positioned within the cell. Sister replisomes separate and move to opposite cell halves shortly after initiation, migrating outwards as replication proceeds and both returning to midcell as replication termination approaches. DNA polymerase is maintained at stalled replication forks, and over short intervals of time replisomes are more dynamic than genetic loci. The data are inconsistent with models in which replisomes associated with sister forks act within a fixed replication factory. We conclude that independent replication forks follow the path of the compacted chromosomal DNA, with no structure other than DNA anchoring the replisome to any particular cellular region.
PMCID: PMC2288635  PMID: 18394992
9.  MukB colocalizes with the oriC region and is required for organization of the two Escherichia coli chromosome arms into separate cell halves 
Molecular Microbiology  2007;65(6):1485-1492.
The circular Escherichia coli chromosome is organized by bidirectional replication into two equal left and right arms (replichores). Each arm occupies a separate cell half, with the origin of replication (oriC) at mid-cell. E. coli MukBEF belongs to the ubiquitous family of SMC protein complexes that play key roles in chromosome organization and processing. In mukBEF mutants, viability is restricted to low temperature with production of anucleate cells, reflecting chromosome segregation defects. We show that in mukB mutant cells, the two chromosome arms do not separate into distinct cell halves, but extend from pole to pole with the oriC region located at the old pole. Mutations in topA, encoding topoisomerase I, do not suppress the aberrant positioning of chromosomal loci in mukB cells, despite suppressing the temperature-sensitivity and production of anucleate cells. Furthermore, we show that MukB and the oriC region generally colocalize throughout the cell cycle, even when oriC localization is aberrant. We propose that MukBEF initiates the normal bidirectional organization of the chromosome from the oriC region.
PMCID: PMC2169520  PMID: 17824928
10.  Replication-directed sister chromosome alignment in Escherichia coli 
Molecular Microbiology  2009;75(5):1090-1097.
Non-replicating Escherichia coli chromosomes are organized as sausage-shaped structures with the left (L) and the right (R) chromosome arms (replichores) on opposite cell halves and the replication origin (oriC) close to midcell. The replication termination region (ter) therefore passes between the two outer edges of the nucleoid. Four alignment patterns of the two sister chromosomes within a cell have been detected in an asynchronous population, with the pattern predominating. We test the hypothesis that the minority and patterns arise because of pausing of DNA replication on the right and left replichores respectively. The data resulting from transient pausing or longer-term site-specific blocking of replication show that paused/blocked loci remain close to midcell and the normally replicated-segregated loci locate to the outer regions of the nucleoid, therefore providing experimental support for a direct mechanistic link between DNA replication and chromosome organization.
PMCID: PMC2859247  PMID: 20487299

Results 1-10 (10)