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1.  The SMC Complex MukBEF Recruits Topoisomerase IV to the Origin of Replication Region in Live Escherichia coli 
mBio  2014;5(1):e01001-13.
The Escherichia coli structural maintenance of chromosome (SMC) complex, MukBEF, and topoisomerase IV (TopoIV) interact in vitro through a direct contact between the MukB dimerization hinge and the C-terminal domain of ParC, the catalytic subunit of TopoIV. The interaction stimulates catalysis by TopoIV in vitro. Using live-cell quantitative imaging, we show that MukBEF directs TopoIV to ori, with fluorescent fusions of ParC and ParE both forming cellular foci that colocalize with those formed by MukBEF throughout the cell cycle and in cells unable to initiate DNA replication. Removal of MukBEF leads to loss of fluorescent ParC/ParE foci. In the absence of functional TopoIV, MukBEF forms multiple foci that are distributed uniformly throughout the nucleoid, whereas multiple catenated oris cluster at midcell. Once functional TopoIV is restored, the decatenated oris segregate to positions that are largely coincident with the MukBEF foci, thereby providing support for a mechanism by which MukBEF acts in chromosome segregation by positioning newly replicated and decatenated oris. Additional evidence for such a mechanism comes from the observation that in TopoIV-positive (TopoIV+) cells, newly replicated oris segregate rapidly to the positions of MukBEF foci. Taken together, the data implicate MukBEF as a key component of the DNA segregation process by acting in concert with TopoIV to promote decatenation and positioning of newly replicated oris.
Mechanistic understanding of how newly replicated bacterial chromosomes are segregated prior to cell division is incomplete. In this work, we provide in vivo experimental support for the view that topoisomerase IV (TopoIV), which decatenates newly replicated sister duplexes as a prelude to successful segregation, is directed to the replication origin region of the Escherichia coli chromosome by the SMC (structural maintenance of chromosome) complex, MukBEF. We provide in vivo data that support the demonstration in vitro that the MukB interaction with TopoIV stimulates catalysis by TopoIV. Finally, we show that MukBEF directs the normal positioning of sister origins after their replication and during their segregation. Overall, the data support models in which the coordinate and sequential action of TopoIV and MukBEF plays an important role during bacterial chromosome segregation.
PMCID: PMC3950513  PMID: 24520061
2.  MinC, MinD, and MinE Drive Counter-oscillation of Early-Cell-Division Proteins Prior to Escherichia coli Septum Formation 
mBio  2013;4(6):e00856-13.
Bacterial cell division initiates with the formation of a ring-like structure at the cell center composed of the tubulin homolog FtsZ (the Z-ring), which acts as a scaffold for the assembly of the cell division complex, the divisome. Previous studies have suggested that the divisome is initially composed of FtsZ polymers stabilized by membrane anchors FtsA and ZipA, which then recruit the remaining division proteins. The MinCDE proteins prevent the formation of the Z-ring at poles by oscillating from pole to pole, thereby ensuring that the concentration of the Z-ring inhibitor, MinC, is lowest at the cell center. We show that prior to septum formation, the early-division proteins ZipA, ZapA, and ZapB, along with FtsZ, assemble into complexes that counter-oscillate with respect to MinC, and with the same period. We propose that FtsZ molecules distal from high concentrations of MinC form relatively slowly diffusing filaments that are bound by ZapAB and targeted to the inner membrane by ZipA or FtsA. These complexes may facilitate the early stages of divisome assembly at midcell. As MinC oscillates toward these complexes, FtsZ oligomerization and bundling are inhibited, leading to shorter or monomeric FtsZ complexes, which become less visible by epifluorescence microscopy because of their rapid diffusion. Reconstitution of FtsZ-Min waves on lipid bilayers shows that FtsZ bundles partition away from high concentrations of MinC and that ZapA appears to protect FtsZ from MinC by inhibiting FtsZ turnover.
A big issue in biology for the past 100 years has been that of how a cell finds its middle. In Escherichia coli, over 20 proteins assemble at the cell center at the time of division. We show that the MinCDE proteins, which prevent the formation of septa at the cell pole by inhibiting FtsZ, drive the counter-oscillation of early-cell-division proteins ZapA, ZapB, and ZipA, along with FtsZ. We propose that FtsZ forms filaments at the pole where the MinC concentration is the lowest and acts as a scaffold for binding of ZapA, ZapB, and ZipA: such complexes are disassembled by MinC and reform within the MinC oscillation period before accumulating at the cell center at the time of division. The ability of FtsZ to be targeted to the cell center in the form of oligomers bound by ZipA and ZapAB may facilitate the early stages of divisome assembly.
PMCID: PMC3870257  PMID: 24327341
3.  The N-Terminal Membrane-Spanning Domain of the Escherichia coli DNA Translocase FtsK Hexamerizes at Midcell 
mBio  2013;4(6):e00800-13.
Bacterial FtsK plays a key role in coordinating cell division with the late stages of chromosome segregation. The N-terminal membrane-spanning domain of FtsK is required for cell division, whereas the C-terminal domain is a fast double-stranded DNA (dsDNA) translocase that brings the replication termination region of the chromosome to midcell, where it facilitates chromosome unlinking by activating XerCD-dif site-specific recombination. Therefore, FtsK coordinates the late stages of chromosome segregation with cell division. Although the translocase is known to act as a hexamer on DNA, it is unknown when and how hexamers form, as is the number of FtsK molecules in the cell and within the divisome. Using single-molecule live-cell imaging, we show that newborn Escherichia coli cells growing in minimal medium contain ~40 membrane-bound FtsK molecules that are largely monomeric; the numbers increase proportionately with cell growth. After recruitment to the midcell, FtsK is present only as hexamers. Hexamers are observed in all cells and form before any visible sign of cell constriction. An average of 7 FtsK hexamers per cell are present at midcell, with the N-terminal domain being able to hexamerize independently of the translocase. Detergent-solubilized and purified FtsK N-terminal domains readily form hexamers, as determined by in vitro biochemistry, thereby supporting the in vivo data. The hexameric state of the FtsK N-terminal domain at the division site may facilitate assembly of a functional C-terminal DNA translocase on chromosomal DNA.
In the rod-shaped bacterium Escherichia coli, more than a dozen proteins act at the cell center to mediate cell division, which initiates while chromosome replication and segregation are under way. The protein FtsK coordinates cell division with the late stages of chromosome segregation. The N-terminal part of FtsK is membrane embedded and acts in division, while the C-terminal part forms a hexameric ring on chromosomal DNA, which the DNA can translocate rapidly to finalize chromosome segregation. Using quantitative live-cell imaging, which measures the position and number of FtsK molecules, we show that in all cells, FtsK hexamers form only at the cell center at the initiation of cell division. Furthermore, the FtsK N-terminal portion forms hexamers independently of the C-terminal translocase.
PMCID: PMC3870252  PMID: 24302254
4.  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
5.  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
6.  Genome organization and regulation 
PMCID: PMC3596235  PMID: 23486399
7.  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
8.  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
9.  Activation of XerCD-dif recombination by the FtsK DNA translocase 
Nucleic Acids Research  2011;39(12):5140-5148.
The FtsK translocase pumps dsDNA directionally at ∼5 kb/s and facilitates chromosome unlinking by activating XerCD site-specific recombination at dif, located in the replication terminus of the Escherichia coli chromosome. We show directly that the γ regulatory subdomain of FtsK activates XerD catalytic activity to generate Holliday junction intermediates that can then be resolved by XerC. Furthermore, we demonstrate that γ can activate XerCD-dif recombination in the absence of the translocase domain, when it is fused to XerCD, or added in isolation. In these cases the recombination products are topologically complex and would impair chromosome unlinking. We propose that FtsK translocation and activation of unlinking are normally coupled, with the translocation being essential for ensuring that the products of recombination are topologically unlinked, an essential feature of the role of FtsK in chromosome segregation.
PMCID: PMC3130261  PMID: 21371996
10.  A streptavidin variant with slower biotin dissociation and increased mechanostability 
Nature methods  2010;7(5):391-393.
Streptavidin binds biotin-conjugates with exceptional stability, but dissociation does occur and can be limiting in imaging, DNA amplification, and nanotechnology. We identified a mutant streptavidin, which we call traptavidin, showing ~10-fold slower biotin off-rate, increased mechanical strength, and improved thermostability; this resilience should find diverse applications. We show that the motor protein FtsK could strip proteins from DNA, rapidly displacing streptavidin from biotinylated DNA; traptavidin resisted displacement and thus indicated the force generated by FtsK translocation.
PMCID: PMC2862113  PMID: 20383133
11.  Independent Segregation of the Two Arms of the Escherichia coli ori Region Requires neither RNA Synthesis nor MreB Dynamics ▿ § ‡  
Journal of Bacteriology  2010;192(23):6143-6153.
The mechanism of Escherichia coli chromosome segregation remains elusive. We present results on the simultaneous tracking of segregation of multiple loci in the ori region of the chromosome in cells growing under conditions in which a single round of replication is initiated and completed in the same generation. Loci segregated as expected for progressive replication-segregation from oriC, with markers placed symmetrically on either side of oriC segregating to opposite cell halves at the same time, showing that sister locus cohesion in the origin region is local rather than extensive. We were unable to observe any influence on segregation of the proposed centromeric site, migS, or indeed any other potential cis-acting element on either replication arm (replichore) in the AB1157 genetic background. Site-specific inhibition of replication close to oriC on one replichore did not prevent segregation of loci on the other replichore. Inhibition of RNA synthesis and inhibition of the dynamic polymerization of the actin homolog MreB did not affect ori and bulk chromosome segregation.
PMCID: PMC2981198  PMID: 20889756
12.  fpr, a Deficient Xer Recombination Site from a Salmonella Plasmid, Fails To Confer Stability by Dimer Resolution: Comparative Studies with the pJHCMW1 mwr Site▿  
Journal of Bacteriology  2009;192(3):883-887.
Salmonella plasmid pFPTB1 includes a Tn3-like transposon and a Xer recombination site, fpr, which mediates site-specific recombination at efficiencies lower than those required for stabilizing a plasmid by dimer resolution. Mutagenesis and comparative studies with mwr, a site closely related to fpr, indicate that there is an interdependence of the sequences in the XerC binding region and the central region in Xer site-specific recombination sites.
PMCID: PMC2812456  PMID: 19966005
13.  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
14.  Separating speed and ability to displace roadblocks during DNA translocation by FtsK 
The EMBO Journal  2010;29(8):1423-1433.
FtsK translocates dsDNA directionally at >5 kb/s, even under strong forces. In vivo, the action of FtsK at the bacterial division septum is required to complete the final stages of chromosome unlinking and segregation. Despite the availability of translocase structures, the mechanism by which ATP hydrolysis is coupled to DNA translocation is not understood. Here, we use covalently linked translocase subunits to gain insight into the DNA translocation mechanism. Covalent trimers of wild-type subunits dimerized efficiently to form hexamers with high translocation activity and an ability to activate XerCD-dif chromosome unlinking. Covalent trimers with a catalytic mutation in the central subunit formed hexamers with two mutated subunits that had robust ATPase activity. They showed wild-type translocation velocity in single-molecule experiments, activated translocation-dependent chromosome unlinking, but had an impaired ability to displace either a triplex oligonucleotide, or streptavidin linked to biotin-DNA, during translocation along DNA. This separation of translocation velocity and ability to displace roadblocks is more consistent with a sequential escort mechanism than stochastic, hand-off, or concerted mechanisms.
PMCID: PMC2868570  PMID: 20379135
Escherichia coli chromosome segregation; FtsK DNA translocase; molecular motor
15.  FtsK translocation on DNA stops at XerCD-dif 
Nucleic Acids Research  2009;38(1):72-81.
Escherichia coli FtsK is a powerful, fast, double-stranded DNA translocase, which can strip proteins from DNA. FtsK acts in the late stages of chromosome segregation by facilitating sister chromosome unlinking at the division septum. KOPS-guided DNA translocation directs FtsK towards dif, located within the replication terminus region, ter, where FtsK activates XerCD site-specific recombination. Here we show that FtsK translocation stops specifically at XerCD-dif, thereby preventing removal of XerCD from dif and allowing activation of chromosome unlinking by recombination. Stoppage of translocation at XerCD-dif is accompanied by a reduction in FtsK ATPase and is not associated with FtsK dissociation from DNA. Specific stoppage at recombinase-DNA complexes does not require the FtsKγ regulatory subdomain, which interacts with XerD, and is not dependent on either recombinase-mediated DNA cleavage activity, or the formation of synaptic complexes.
PMCID: PMC2800217  PMID: 19854947
16.  KOPS-guided DNA translocation by FtsK safeguards Escherichia coli chromosome segregation 
Molecular Microbiology  2008;71(4):1031-1042.
The septum-located DNA translocase, FtsK, acts to co-ordinate the late steps of Escherichia coli chromosome segregation with cell division. The FtsK γ regulatory subdomain interacts with 8 bp KOPS DNA sequences, which are oriented from the replication origin to the terminus region (ter) in each arm of the chromosome. This interaction directs FtsK translocation towards ter where the final chromosome unlinking by decatenation and chromosome dimer resolution occurs. Chromosome dimer resolution requires FtsK translocation along DNA and its interaction with the XerCD recombinase bound to the recombination site, dif, located within ter. The frequency of chromosome dimer formation is ∼15% per generation in wild-type cells. Here we characterize FtsK alleles that no longer recognize KOPS, yet are proficient for translocation and chromosome dimer resolution. Non-directed FtsK translocation leads to a small reduction in fitness in otherwise normal cell populations, as a consequence of ∼70% of chromosome dimers being resolved to monomers. More serious consequences arise when chromosome dimer formation is increased, or their resolution efficiency is impaired because of defects in chromosome organization and processing. For example, when Cre–loxP recombination replaces XerCD–dif recombination in dimer resolution, when functional MukBEF is absent, or when replication terminates away from ter.
PMCID: PMC2680272  PMID: 19170870
17.  mwr Xer site-specific recombination is hypersensitive to DNA supercoiling 
Nucleic Acids Research  2009;37(11):3580-3587.
The multiresistance plasmid pJHCMW1, first identified in a Klebsiella pneumoniae strain isolated from a neonate with meningitis, includes a Xer recombination site, mwr, with unique characteristics. Efficiency of resolution of mwr-containing plasmid dimers is strongly dependent on the osmotic pressure of the growth medium. An increase in supercoiling density of plasmid DNA was observed as the osmotic pressure of the growth culture decreased. Reporter plasmids containing directly repeated mwr, or the related cer sites were used to test if DNA topological changes were correlated with significant changes in efficiency of Xer recombination. Quantification of Holliday junctions showed that while recombination at cer was efficient at all levels of negative supercoiling, recombination at mwr became markedly less efficient as the level of supercoiling was reduced. These results support a model in which modifications at the level of supercoiling density caused by changes in the osmotic pressure of the culture medium affects resolution of mwr-containing plasmid dimers, a property that separates mwr from other Xer recombination target sites.
PMCID: PMC2699498  PMID: 19359357
18.  Molecular Mechanism of Sequence-Directed DNA Loading and Translocation by FtsK 
Molecular cell  2008;31(4):498-509.
Dimeric circular chromosomes, formed by recombination between monomer sisters, cannot be segregated to daughter cells at cell division. XerCD site-specific recombination at the Escherichia coli dif site converts these dimers to monomers in a reaction that requires the DNA translocase FtsK. Short DNA sequences, KOPS (GGGNAGGG), which are polarized toward dif in the chromosome, direct FtsK translocation. FtsK interacts with KOPS through a C-terminal winged helix domain γ. The crystal structure of three FtsKγ domains bound to 8 bp KOPS DNA demonstrates how three γ domains recognize KOPS. Using covalently linked dimers of FtsK, we infer that three γ domains per hexamer are sufficient to recognize KOPS and load FtsK and subsequently activate recombination at dif. During translocation, FtsK fails to recognize an inverted KOPS sequence. Therefore, we propose that KOPS act solely as a loading site for FtsK, resulting in a uni-directionally oriented hexameric motor upon DNA.
PMCID: PMC2570039  PMID: 18722176
19.  The FtsK γ domain directs oriented DNA translocation by interacting with KOPS 
The bacterial septum-located DNA translocase FtsK coordinates circular chromosome segregation with cell division. Rapid translocation of DNA by FtsK is directed by 8-base-pair DNA motifs (KOPS), so that newly replicated termini are brought together at the developing septum, thereby facilitating completion of chromosome segregation. Translocase functions reside in three domains, α, β and γ. FtsKαβ are necessary and sufficient for ATP hydrolysis–dependent DNA translocation, which is modulated by FtsKγ through its interaction with KOPS. By solving the FtsKγ structure by NMR, we show that γ is a winged-helix domain. NMR chemical shift mapping localizes the DNA-binding site on the γ domain. Mutated proteins with substitutions in the FtsKγ DNA-recognition helix are impaired in DNA binding and KOPS recognition, yet remain competent in DNA translocation and XerCD-dif site-specific recombination, which facilitates the late stages of chromosome segregation.
PMCID: PMC2556771  PMID: 17057717
20.  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
21.  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
22.  Sublethal Concentrations of the Aminoglycoside Amikacin Interfere with Cell Division without Affecting Chromosome Dynamics▿  
Aminoglycosides bind to the 16S rRNA at the tRNA acceptor site (A site) and disturb protein synthesis by inducing codon misreading. We investigated Escherichia coli cell elongation and division, as well as the dynamics of chromosome replication and segregation, in the presence of sublethal concentrations of amikacin (AMK). The fates of the chromosome ori and ter loci were monitored by visualization by using derivatives of LacI and TetR fused to fluorescent proteins in E. coli strains that carry operator arrays at the appropriate locations. The results showed that cultures containing sublethal concentrations of AMK contained abnormally elongated cells. The chromosomes in these cells were properly located, suggesting that the dynamics of replication and segregation were normal. FtsZ, an essential protein in the process of cell division, was studied by using an ectopic FtsZ-cyan fluorescent protein fusion. Consistent with a defect in cell division, we revealed that the Z ring failed to properly assemble in these elongated cells.
PMCID: PMC1797645  PMID: 17043119
23.  Replication fork blockage by transcription factor-DNA complexes in Escherichia coli 
Nucleic Acids Research  2006;34(18):5194-5202.
All organisms require mechanisms that resuscitate replication forks when they break down, reflecting the complex intracellular environments within which DNA replication occurs. Here we show that as few as three lac repressor-operator complexes block Escherichia coli replication forks in vitro regardless of the topological state of the DNA. Blockage with tandem repressor-operator complexes was also observed in vivo, demonstrating that replisomes have a limited ability to translocate through high affinity protein–DNA complexes. However, cells could tolerate tandem repressor-bound operators within the chromosome that were sufficient to block all forks in vitro. This discrepancy between in vitro and in vivo observations was at least partly explained by the ability of RecA, RecBCD and RecG to abrogate the effects of repressor-operator complexes on cell viability. However, neither RuvABC nor RecF were needed for normal cell growth in the face of such complexes. Holliday junction resolution by RuvABC and facilitated loading of RecA by RecF were not therefore critical for tolerance of protein–DNA blocks. We conclude that there is a trade-off between efficient genome duplication and other aspects of DNA metabolism such as transcriptional control, and that recombination enzymes, either directly or indirectly, provide the means to tolerate such conflicts.
PMCID: PMC1636447  PMID: 17000639
24.  Differences in Resolution of mwr-Containing Plasmid Dimers Mediated by the Klebsiella pneumoniae and Escherichia coli XerC Recombinases: Potential Implications in Dissemination of Antibiotic Resistance Genes 
Journal of Bacteriology  2006;188(8):2812-2820.
Xer-mediated dimer resolution at the mwr site of the multiresistance plasmid pJHCMW1 is osmoregulated in Escherichia coli containing either the Escherichia coli Xer recombination machinery or Xer recombination elements from K. pneumoniae. In the presence of K. pneumoniae XerC (XerCKp), the efficiency of recombination is lower than that in the presence of the E. coli XerC (XerCEc) and the level of dimer resolution is insufficient to stabilize the plasmid, even at low osmolarity. This lower efficiency of recombination at mwr is observed in the presence of E. coli or K. pneumoniae XerD proteins. Mutagenesis experiments identified a region near the N terminus of XerCKp responsible for the lower level of recombination catalyzed by XerCKp at mwr. This region encompasses the second half of the predicted α-helix B and the beginning of the predicted α-helix C. The efficiencies of recombination at other sites such as dif or cer in the presence of XerCKp or XerCEc are comparable. Therefore, XerCKp is an active recombinase whose action is impaired on the mwr recombination site. This characteristic may result in restriction of the host range of plasmids carrying this site, a phenomenon that may have important implications in the dissemination of antibiotic resistance genes.
PMCID: PMC1446988  PMID: 16585742
25.  Dissection of a functional interaction between the DNA translocase, FtsK, and the XerD recombinase 
Molecular Microbiology  2006;59(6):1754-1766.
Successful bacterial circular chromosome segregation requires that any dimeric chromosomes, which arise by crossing over during homologous recombination, are converted to monomers. Resolution of dimers to monomers requires the action of the XerCD site-specific recombinase at dif in the chromosome replication terminus region. This reaction requires the DNA translocase, FtsKC, which activates dimer resolution by catalysing an ATP hydrolysis-dependent switch in the catalytic state of the nucleoprotein recombination complex. We show that a 62-amino-acid fragment of FtsKC interacts directly with the XerD C-terminus in order to stimulate the cleavage by XerD of BSN, a dif-DNA suicide substrate containing a nick in the ‘bottom’ strand. The resulting recombinase–DNA covalent complex can undergo strand exchange with intact duplex dif in the absence of ATP. FtsKC-mediated stimulation of BSN cleavage by XerD requires synaptic complex formation. Mutational impairment of the XerD–FtsKC interaction leads to reduction in the in vitro stimulation of BSN cleavage by XerD and a concomitant deficiency in the resolution of chromosomal dimers at dif in vivo, although other XerD functions are not affected.
PMCID: PMC1413583  PMID: 16553881

Results 1-25 (36)