Initiating bacterial mitosis: Understanding the mechanism of ParA-mediated chromosome segregation

Comment on: Ptacin JL, et al. Nat Cell Biol 2010; 12:791–8.     

The bacterial nucleoid revisited.

This review compares the results of different methods of investigating the morphology of nucleoids of bacteria grown under conditions favoring short generation times. We consider the evidence from fixed and stained specimens, from phase-contrast and fluorescence microscopy of growing bacteria, and from electron microscopy of whole as well as thinly sectioned ones. It is concluded that the nucleoid of growing cells is in a dynamic state: part of the chromatin is "pulled out" of the bulk of the nucleoid in order to be transcribed. This activity is performed by excrescences which extend far into the cytoplasm so as to reach the maximum of available ribosomes. Different means of fixation provide markedly different views of the texture of the DNA-containing plasm of the bulk of the nucleoid. Conventional chemical fixatives stabilize the cytoplasm of bacteria but not their protein-low chromatin. Uranyl acetate does cross-link the latter well but only if the cytoplasm has first been fixed conventionally. In the interval between the two fixations, the DNA arranges itself in liquid-crystalline form, supposedly because of loss of supercoiling. In stark contrast, cryofixation preserves bacterial chromatin in a finely granular form, believed to reflect its native strongly negatively supercoiled state. In dinoflagellates the DNA of their permanently visible chromosomes (also low in histone-like protein) is natively present as a liquid crystal. The arrangement of chromatin in Epulocystis fishelsoni, one of the largest known prokaryotes, is briefly described.     

Delayed nucleoid segregation in Escherichia coli

To study the role of cell division in the process of nucleoid segregation, we measured the DNA content of individual nucleoids in isogenic Escherichia coli cell division mutants by image cytometry. In pbpB(Ts) and ftsZ strains growing as filaments at 42 degrees C, nucleoids contained, on average, more than two chromosome equivalents compared with 1.6 in wild-type cells. Because similar results were obtained with a pbpB recA strain, the increased DNA content cannot be ascribed to the occurrence of chromosome dimers. From the determination of the amount of DNA per cell and per individual nucleoid after rifampicin inhibition, we estimated the C and D periods (duration of a round of replication and time between termination and cell division respectively), as well as the D' period (time between termination and nucleoid separation). Compared with the parent strain and in contrast to ftsQ, ftsA and ftsZ mutants, pbpB(Ts) cells growing at the permissive temperature (28 degrees C) showed a long D' period (42 min versus 18 min in the parent) indicative of an extended segregation time. The results indicate that a defective cell division protein such as PbpB not only affects the division process but also plays a role in the last stage of DNA segregation. We propose that PbpB is involved in the assembly of the divisome and that this structure enhances nucleoid segregation.     

Diversity and redundancy in bacterial chromosome segregation mechanisms

Bacterial cells are much smaller and have a much simpler overall structure and organization than eukaryotes. Several prominent differences in cell organization are relevant to the mechanisms of chromosome segregation, particularly the lack of an overt chromosome condensation/decondensation cycle and the lack of a microtubule-based spindle. Although bacterial chromosomes have a rather dispersed appearance, they nevertheless have an underlying high level of spatial organization. During the DNA replication cycle, early replicated (oriC) regions are localized towards the cell poles, whereas the late replicated terminus (terC) region is medially located. This spatial organization is thought to be driven by an active segregation mechanism that separates the sister chromosomes continuously as replication proceeds. Comparisons of various well-characterized bacteria suggest that the mechanisms of chromosome segregation are likely to be diverse, and that in many bacteria, multiple overlapping mechanisms may contribute to efficient segregation. One system in which the molecular mechanisms of chromosome segregation are beginning to be elucidated is that of sporulating cells of Bacillus subtilis. The key components of this system have been identified, and their functions are understood, in outline. Although this system appears to be specialized, most of the functions are conserved widely throughout the bacteria.     

A locus affecting nucleoid segregation in Salmonella typhimurium.

Thirteen temperature-sensitive lethal mutations of Salmonella typhimurium map near metC at 65 min and form the clmF (conditional lethal mutation) locus. The mutations in this region were ordered by three-point transduction crosses. After a shift to the nonpermissive temperature, many of these clmF mutants failed to complete the segregation of nucleoids into daughter cells; daughter nucleoids appeared incompletely separated and asymmetrically positioned within cells. Some clmF mutants showed instability of F' episomes at permissive growth temperatures yet showed no detectable defect with smaller multicopy plasmids such as pSC101 or pBR322. In addition, many of the clmF mutants rapidly lost viability yet continued DNA replication at the nonpermissive temperature. These results suggest that the clmF locus encodes at least one indispensable gene product that is required for faithful partitioning of the bacterial nucleoid and F-plasmid replicons.     

SMC complexes in bacterial chromosome condensation and segregation

Bacterial chromosomes segregate via a partition apparatus that employs a score of specialized proteins. The SMC complexes play a crucial role in the chromosome partitioning process by organizing bacterial chromosomes through their ATP-dependent chromatin-compacting activity. Recent progress in the composition of these complexes and elucidation of their structural and enzymatic properties has advanced our comprehension of chromosome condensation and segregation mechanics in bacteria.     

Basic mechanism of eukaryotic chromosome segregation

We now have firm evidence that the basic mechanism of chromosome segregation is similar among diverse eukaryotes as the same genes are employed. Even in prokaryotes, the very basic feature of chromosome segregation has similarities to that of eukaryotes. Many aspects of chromosome segregation are closely related to a cell cycle control that includes stage-specific protein modification and proteolysis. Destruction of mitotic cyclin and securin leads to mitotic exit and separase activation, respectively. Key players in chromosome segregation are SMC-containing cohesin and condensin, DNA topoisomerase II, APC/C ubiquitin ligase, securin-separase complex, aurora passengers, and kinetochore microtubule destabilizers or regulators. In addition, the formation of mitotic kinetochore and spindle apparatus is absolutely essential. The roles of principal players in basic chromosome segregation are discussed: most players have interphase as well as mitotic functions. A view on how the centromere/kinetochore is formed is described.     

Coordination of cell division and chromosome segregation by a nucleoid occlusion protein in Bacillus subtilis

A range of genetical and physiological experiments have established that diverse bacterial cells possess a function called nucleoid occlusion, which acts to prevent cell division in the vicinity of the nucleoid. We have identified a specific effector of nucleoid occlusion in Bacillus subtilis, Noc (YyaA), as an inhibitor of division that is also a nonspecific DNA binding protein. Under various conditions in which the cell cycle is perturbed, Noc prevents the division machinery from assembling in the vicinity of the nucleoid. Unexpectedly, cells lacking both Noc and the Min system (which prevents division close to the cell poles) are blocked for division, apparently because they establish multiple nonproductive accumulations of division proteins. The results help to explain how B. subtilis specifies the division site under a range of conditions and how it avoids catastrophic breakage of the chromosome by division through the nucleoid.     

The bacterial cell cycle: DNA replication, nucleoid segregation, and cell division

Data on the bacterial cell cycle published in the last 10-15 years are considered, with a special stress on studies of nucleoid segregation between dividing cells. The degree of similarity between the eukaryotic mitotic apparatus and the apparatus performing nucleoid separation is discussed.     

Coralline shape of the bacterial nucleoid after cryofixation.

A new procedure of immunostaining sections of cryofixed and freeze-substituted Escherichia coli shows that DNA extends from its bulk into small ribosome-free spaces throughout the cytoplasm, resulting in a coralline-shaped nucleoid. Low-resolution imaging of a bacterium reconstructed from serial sections demonstrated that the small excrescencies are not resolved. The resulting photograph shows the same features as phase-contrast light micrographs.     

Prokaryotic ParA-ParB-parS system links bacterial chromosome segregation with the cell cycle

While the essential role of episomal par loci in plasmid DNA partitioning has long been appreciated, the function of chromosomally encoded par loci is less clear. The chromosomal parA-parB genes are conserved throughout the bacterial kingdom and encode proteins homologous to those of the plasmidic Type I active partitioning systems. The third conserved element, the centromere-like sequence called parS, occurs in several copies in the chromosome. Recent studies show that the ParA-ParB-parS system is a key player of a mitosis-like process ensuring proper intracellular localization of certain chromosomal regions such as oriC domain and their active and directed segregation. Moreover, the chromosomal par systems link chromosome segregation with initiation of DNA replication and the cell cycle.     

A cluster of genes that affects nucleoid segregation in Salmonella typhimurium.

Thirteen conditional lethal mutations in genes of Salmonella typhimurium map at the clmF locus and affect both viability and the faithful partitioning of daughter nucleoids. These mutations have now been divided into three complementation groups by using cloned fragments of S. typhimurium DNA and renamed parC, parE, and parF. The proteins produced from the cloned fragments predict that ParC is an 85-kD protein, ParE is 75 kD in size, and ParF, 27 kD. The parE gene is about 5 kb upstream of the parC gene, and parC is just upstream of parF. Genes situated between parC and parE produce at least two proteins of unknown function. The DNA sequence of the S. typhimurium parC gene was determined and has 56% homology with the first 1400 base pairs of the Escherichia coli gryA gene, which encodes the A subunit of DNA gyrase, and 85% homology with the E. coli parC gene. Despite the strong homology between gryA and parC, these two genes cannot substitute for one another. The DNA sequence of the S. typhimurium parF gene was determined and predicts a protein with a hydrophobic N terminus. The ParF protein may interact with ParC and ParE to anchor these proteins to the membrane. These results raise questions about the relative roles of gyrase and ParCEF in nucleoid decatenation. In addition, the parC and gyrA genes provide an example of the evolution of essential functions by gene duplication.     

FtsZ and nucleoid segregation during outgrowth of Bacillus subtilis spores.

Spores of a strain of Bacillus subtilis in which ftsZ was under the control of the spac promoter were allowed to germinate and grow out in the presence of increasing concentrations of isopropyl-beta-D-thiogalactopyranoside (IPTG). Over the IPTG concentration range of 0 to 10(-3) M, the level of FtsZ from the time when the first nucleoid segregations were occurring, measured in Western blot (immunoblot) transfer experiments, varied between 15 and 100% of that in the wild type. Septation was completely blocked (for at least several hours) when the amount of FtsZ was < 30% of the wild-type level. At all levels of ftsZ induction, the timing and rate of segregation of nucleoids following the first round of replication were unaltered. It is concluded that FtsZ has no direct role in nucleoid segregation in this situation.     

Transmission electron microscopy of the bacterial nucleoid

Water-containing biological material cannot withstand the vacuum of the transmission electron microscope. The classical solution to this problem has been to dehydrate chemically fixed biological samples and then embed them in resin. During such treatment, the bacterial nucleoid is especially prone to aggregation, which affects its global shape and fine structure. Initial attempts to deal with aggregation by optimizing chemical fixation yielded contradictory results. Two decades ago, the situation improved with the introduction of freeze-substitution. This method is based on dehydration of unfixed cryo-immobilized samples at low temperature, which substantially reduces aggregation. As a result, the global shape of the nucleoid can be fairly well defined. Overall, in actively growing bacteria, the nucleoids are dispersed and "coralline" but become more confined when growth ceases. However, it is usually impossible to determine the molecular arrangement of DNA in the nucleoids of freeze-substituted bacteria because crystallization and the subsequent removal of water during substitution result in unavoidable distortions at the ultrastructural level. Recently, cryo-electron microscopy of vitreous sections has enabled the fully hydrated bacterial nucleoid to be studied close to the native state. Such studies have revealed aspects of bacterial nucleoid organization that are not preserved by freeze-substitution, including locally parallel or twisted bundles of DNA filaments, which are more frequently observed once bacterial growth has stopped, whereas in actively growing bacteria, the DNA is seen to be in a mostly disordered pattern.