To enable a macromolecule this large to fit within the bacterium, histone-like proteins bind to the DNA, segregating the DNA molecule into around 50 chromosomal domains and making it more compact. In actively growing bacteria, projections of the nucleoid extend into the cytoplasm.
Supercoils are both inserted and removed by topoisomerases. DNA topoisomerases are, therefore, essential in the unwinding, replication, and rewinding of the circular, supercoiled bacterial DNA.
For example, a topoisomerase called DNA gyrase catalyzes the negative supercoiling of the circular DNA found in bacteria. Topoisomerase IV, on the other hand, is involved in the relaxation of the supercoiled circular DNA, enabling the separation of the interlinked daughter chromosomes at the end of bacterial DNA replication.
In general, DNA is replicated by uncoiling of the helix, strand separation by breaking of the hydrogen bonds between the complementary strands, and synthesis of two new strands by complementary base pairing. Replication begins at a specific site in the DNA called the origin of replication ori C.
DNA replication is bidirectional from the origin of replication. To begin DNA replication, unwinding enzymes called DNA helicases cause short segments of the two parent DNA strands to unwind and separate from one another at the origin of replication to form two "Y"-shaped replication forks. Single-strand binding proteins bind to the single-stranded regions so the two strands do not rejoin.
Unwinding of the double-stranded helix generates positive supercoils ahead of the replication fork. Enzymes called topoisomerases counteract this by producing breaks in the DNA and then rejoin them to form negative supercoils in order to relieve this stress in the helical molecule during replication. As the strands continue to unwind and separate in both directions around the entire DNA molecule, new complementary strands are produced by the hydrogen bonding of free DNA nucleotides with those on each parent strand.
As the new nucleotides line up opposite each parent strand by hydrogen bonding, enzymes called DNA polymerases join the nucleotides by way of phosphodiester bonds. Actually, the nucleotides lining up by complementary base pairing are deoxynucleotide triphosphates, composed of a nitrogenous base, deoxyribose, and three phosphates. In bacteria, Par proteins function to separate bacterial chromosomes to opposite poles of the cell during cell division. They bind to the origin of replication of the DNA and physically pull or push the chromosomes apart, similar to the mitotic apparatus of eukaryotic cells.
Fts proteins, such as FtsK in the divisome, also help in separating the replicated bacterial chromosome. DNA polymerase enzymes are only able to join the phosphate group at the 5' carbon of a new nucleotide to the hydroxyl OH group of the 3' carbon of a nucleotide already in the chain.
As a result, DNA can only be synthesized in a 5' to 3' direction while copying a parent strand running in a 3' to 5' direction. Each DNA strand has two ends. The two strands are antiparallel, that is they run in opposite directions. However, the other parent strand - the one running 5' to 3' and called the lagging strand - must be copied discontinuously in short fragments Okazaki fragments of around nucleotides each as the DNA unwinds. This occurs, as mentioned above, at the replisome.
The lagging DNA strand loops out from the leading strand and this enables the replisome to move along both strands pulling the DNA through as replication occurs. However, several reports have tracked replication in other organisms, including Bacillus subtilis Lemon and Grossman, ; Migocki et al.
Findings of these studies may help in the construction of fluorescent fusions of replisome components in other bacteria. It is also important to consider alternative N- and C-terminal fusion, as one, or sometimes both, ends of target proteins may be implicated in inter- or intra-molecular interactions.
The sliding clamp Figure 2A is the protein of choice in most studies and both N- and C-terminal fusions proved to be functional in a range of species Kongsuwan et al. In these experiments, choosing another replisome component may be advisable. Monitoring replisome dynamics in strains expressing fusion proteins encoded on an episomal plasmid is not recommended, as plasmid replication is triggered mainly by the same protein components that trigger chromosomal replication.
Fusion with catalytic core subunits Lemon and Grossman, ; Migocki et al. This was shown for M. Thus proteins other than the catalytic core complex may be a better choice for studies of replisome dynamics. When designing a fluorescent fusion for replisome visualization, additional features should be taken into account, especially oligomerization status, fluorescence yield and spectral properties.
Thus, choosing a fluorescent variant with a lower tendency to undergo oligomerization e. Spectral characteristics and brightness are essential, especially when replisomes are localized together with other cellular components e.
Importantly, FP are sensitive to pH and cannot be utilized to analyze anaerobic bacteria, as maturation of the chromophore requires oxygen molecules Shaner et al. Fluorescent fusion proteins are suitable for both qualitative long-term live cell imaging and quantitative analysis. For example, Y-Pet fusion with a variety of replisome subunits was used to quantify the numbers of copies of particular proteins within a replication eye in vivo Reyes-Lamothe et al.
However, most of these variants lacked the properties required for super-resolution imaging. In the latter case, proteins of interest should be fused with photoactivated or photoconvertible proteins. Recently published studies may provide hints regarding single-molecule resolution microscopy of replication complexes Georgescu et al. The fusion of replisome subunits with HaloTag may be an alternative to FP. The advantage of using direct fluorescent ligands e.
Halo ligands are also suitable for high-resolution microscopy. Replication tracking particularly initiation of replication is often accompanied by localization of nascent oriCs Figure 2B. FROS was efficiently used to localize chromosomal loci, including oriC , terminus and other specific loci on both replichores in a variety of species Viollier et al.
However, it is often difficult to insert the large operator arrays into the chromosome, particularly in highly transcribed regions such as oriC Le and Laub, Thus, low levels of repressor should be produced, usually by using inducible promoters. Additionally, tracking oriCs together with replisomes requires delivery of the repressor-FP fusion protein from the chromosomal locus, either as a part of an operator array construct or inserted into an attachment site.
Although FROS may provide invaluable data, its instability is a major drawback. This system uses an intrinsic feature of ParB, its binding to centromere-like parS sequences Wang et al.
Most bacterial species possess the ParAB S chromosome segregation system, except for several well-studied Gammaproteobacteria, including E. Because most chromosomal parS sites are localized proximal to the oriC -proximal regions Livny et al.
This approach has been shown effective in a number of bacteria, including Mycobacterium , M. In bacteria lacking a chromosomal ParAB S system e. Determination of the specific point and subcellular localization at which replication is initiated requires long-term imaging of living cells from several minutes to hours, depending on the bacterial growth rate and the conditions being tested, e. The simplest way to analyze replication at the single-cell level is to spread the cells of the reporter strain on the agar pad a thin agar layer between the microscope slide and the cover glass or on the bottom of solidified medium inside culture dishes Joyce et al.
Although simple and low-cost, this approach is not always applicable e. Microfluidic flow chambers are used for the latter purposes, as well as for rapidly changing culture conditions e. Various microfluidic chips and plates are commercially available from an increasing number of companies, whereas custom made usually PDMS chips are a cost-reducing alternative and also allow for more personalized applications Wang et al.
The architecture of microfluidic chips and plates varies among studies and choosing the right one should be dictated by the specific study purpose and the availability of additional equipment, e. Localization of the replication machinery at the beginning of DNA synthesis is dependent on oriC position, and is therefore connected with the spatial arrangement of the chromosome.
In bacteria having oriC and ter regions positioned at the mid-cell, the intervening chromosomal regions i. Replisomes in the cells exhibiting a left-ori-right configuration are assembled in the mid-cell region of the chromosome. This pattern has been observed in E. During sporulation, however, the B. Positioning of the oriC at the mid-cell of B. SMC can compact large chromosomal regions, and, by interacting with ParB protein, organizes the oriC -proximal regions in B.
After initiation, E. In comparison, B. Replisome positioning in the cell center can be also found in oval-shaped S. Spatial organization of the chromosome entails positioning of the site of replisome assembly. In the ori-ter organized chromosomes, replication is initiated at the cell pole C , at which the oriC region is anchored by specific proteins i. D Subpolar positioning of replisomes has also been observed in the multiploid bacteria S.
OriC region s and replisome s are indicated as violet and green circles, while chromosome is depicted in light blue. Some bacteria, such as M.
Replisomes oscillate in the old-pole-proximal cell half during most of the replication process, but localize closer to the new cell pole prior to termination Trojanowski et al.
A slight asymmetry in mycobacterial replisome positioning is associated with the apical growth mode of these bacteria. As a result of the asymmetric location of oriC , M. Although M.
Bacteria exhibiting complex life cycles often show an ori-ter chromosome orientation Figure 3C. The anchorage of the chromosome at the old cell pole is maintained by the protein PopZ Bowman et al. Similarly, in V. In contrast, the origin ori II of the second, smaller chromosome chrII is located at mid-cell. Replication of V. As a result of the subpolar localization of C. Interestingly, in P. The multiploid and apically growing bacterial species S. Replication of multiple copies of the S.
In the closely related and diploid species C. Fluorescently tagged ParB attaches to the cell poles, suggesting an ori-ter-ter-ori spatial orientation of C. Described differences among bacteria in the positioning of oriC regions during the replication initiation reflect the different modes of chromosome segregation.
Mid-cell replisomes location results in symmetric segregation of oriC s toward the opposite cell poles, while polar and off-center replisome positioning imply asymmetric segregation of the newly replicated oriC regions. Furthermore, polar localization requires the complex system to either anchor oriC directly at the pole e.
Single-cell fluorescence imaging and fluorescence tagging techniques allow researchers to precisely visualize proteins and their complexes inside living bacterial cells in real time.
These techniques revealed that many proteins are targeted to distinct subcellular positions, where they participate in various cellular processes including chromosome replication. Recent studies using advanced live-cell imaging demonstrated that chromosome replication is coordinated with other key steps of the cell cycle, such as chromosome segregation and cell division.
Proteins or protein complexes involved in condensation i. Additionally, other proteins guiding the oriC region have been recently identified.
Interestingly, they vary significantly among different bacteria, e. All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We apologize that numerous original papers could not be cited due to space limitations.
Arias-Cartin, R. Replication fork passage drives asymmetric dynamics of a critical nucleoid-associated protein in Caulobacter. EMBO J. Aves, S. Once the genome is completely replicated, the two circular DNAs separate and the cell divides.
The process is a lot simpler than mitosis or meiosis, because bacteria don't have multiple chromosomes that have to be sorted out correctly to the two daughter cells. Thus, bacteria are able to grow and divide much faster than eukaryotic cells can. Since bacteria are haploid, is there any way that something at all similar to crossing-over can ever occur in bacteria? Figure 8. In , studies conducted by several researchers determined that the Escherichia coli genome was organized into a single, circular chromosome [4].
The evidence for the structure of chromosomal DNA was shown by images achieved through autoradiography , electron microscopy and moving pictures of DNA using fluorescence microscopy. Cairns was the first researcher to obtain an image of the entire chromosome for E. The technique used was autoradiography where the chromosome for E. However, the sizes of the chromosomes were variable and there was a low frequency of circular forms detected. Further experimentation based on Hfr conjugation convincingly demonstrating that E.
The published data of the circular chromosome in E. Therefore, E. The first evidence of multiple chromosomes in bacteria was found in Rhodobacter sphaeroides , a rod-shaped, Gram-negative bacterium Figure 2 [12].
The researchers were able to provide a complete physical map of the R.
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