Bacterial Mitosis: What’s Taking So Long?

by Merry & Elio

More often than not, basic cellular phenomena have been deciphered first in bacteria, and then later elucidated in eukaryotic cells. A glaring exception is mitosis which was first described for eukaryotic cells in the 1880's. The corresponding story for bacterial chromosomes has remained uncharacteristically elusive. Truth is, we don't know much about it, although there has been some piecemeal progress. A review was published in 2006. A recent paper approaches the question by looking at how plasmids are partitioned to the daughter cells during division of their host bacterium.

R1 is a large plasmid (100 Kb) that carries genes for resistance to drugs and heavy metals. Its 2 to 4 copies per cell are reliably partitioned between the progeny cells during cell division. The researchers isolated a three-component system from these cells that acts somewhat like a mitotic spindle when reconstituted in vitro. Those three components are two proteins encoded by the plasmid par operon (ParM and ParR) and a specific centromere-like DNA sequence within the operon (parC).

The ParM protein is a homolog of actin that, given ATP, polymerizes into actin-like filaments. During cell division, bundles of ParM filaments can be seen extending the length of the cell with their ends attached to the segregating plasmid DNA. Binding to parC requires the cooperation of the ParR protein.

For their in vitro studies, the investigators mixed purified parM and parR proteins with parC DNA attached to 350 nm beads. When they looked at an isolated parC bead, they saw filaments forming continuously and growing out from the bead in all directions, resulting in an aster-like structure. Individual filaments grew to an average length of 1.5 microns before experiencing spontaneous catastrophic disassembly. (This property of the filaments is referred to as dynamic instability and plays a vital role according to the authors.) If, instead, the free end of the filaments contact a second parC bead, a bundle of filaments forms connecting the pair of beads. (See Movie #S-4, where parC DNA is red, the parM filaments green.)

(Right) Individual ParM filaments run from bead to bead. Credit: See below.

Red arrows indicate
photobleaching of
parM spindles.
Credit: See below.

Does elongation takes place by addition of ParM at the ends or all along the filament? Growth is at the ends, as can be seen in the figure and in Movie #S-12 where photobleaching was used to make two reference marks on each spindle. As the spindle elongates, the distance between the bleached marks does not change, whereas the distance between each mark and its nearby parC bead increases at a constant rate

The authors hypothesize that the parM filaments that grow out in all directions from one parR/parC complex efficiently search the entire bacterial cell looking for a fellow complex. If they do not meet one, they disassemble and other filaments continue the search. When they do contact a second parR/parC complex, a stable spindle forms and elongates, separating the two plasmid chromosomes. Sounds like what a mitotic spindle does in eukaryotic cells.

An animated picture of this process is seen in Movie #S-17. (Note that this movie is an 11 MB file.)

This is in vitro—a long way from the cell—but it is heartening, nevertheless. A final thought. What is it that makes chromosome segregation in bacteria so hard to pin down? Is it just size that makes a mitotic apparatus imperceptible in bacteria? Or, as was widely considered for a while, is the cell membrane involved? Any thoughts?

Source of Figures: Ethan C. Garner et al. (2007) Reconstitution of DNA Segregation Driven by Assembly of a Prokaryotic Actin Homolog. Science 315: 1270—1274

Comments

[Robert Murray]

Here is a historical note driven by the revelation that there is involvement of a motor actin-like protein in the aggregation of both a bacterial chromosome and accompanying large plasmid. This awakens memories of the 1950's polemic about direct nuclear division versus a mitosis-like chromosomal ballet. It was a matter of intrpretation of images in fixed and stained preparations. Model species (e.g., B. subtilis) showed fairly compact nuclear bodies that somehow formed two similarly shaped bodies during cell division; other model species (e.g., B. megaterium) showed constellations of chromatinic bits in a patch form of nucleus. Two views resulted: C.F. Robinow contended that generally the nuclei behaved as a unit and divided directly while E.D. Delamater contended that the segregation was more likely represented by the patch forms in which select nuclei exemplified stages in the classical mitotic cycle. Each of them had students and supporters and their various interpretations generated heated arguments entertaining enough for cytological papers to be given in the biggest meeting rooms. It went on for years but in the end genetic studies were convincing that the main model species (including by then E. coli) had only a single linkage group. The argument collapsed and there was a formal withdrawal of the concept of a mitosis process in bacteria.
So now two aging, friendly bloggers who were disposed on one side or the other in the 50's are faced with a dilemma. Was each side partially right or partially wrong? Anyway, is this the proper application of the term mitosis, which to some of us is the descriptive term for a choreographed ballet of eucaryotic chromosomes assisted by centrioles and spindle structures? Maybe it does not matter: they move anyway and the mechanism has at least one element that reminds us of how mitosis works.

[Conrad Woldringh]

Bacterial Mitosis: What's Taking So Long? Those who agree with this question have to exert more patience for two reasons:
First, because, in my view, there is no such simple thing as "bacterial mitosis". The term "mitotic-like segregation" is used, some microbiologists told me, because the terminology of G1, S, G2 and M phases makes it easier to publish in Eukaryotic-Cell-like journals.
The second reason is that the eukaryotic process of strand resolution within the cohesin/condensin complexes, with which the process of bacterial segregation can better be compared, is far from understood. In a review of 2002 ("Segregating sister genomes: the molecular biology of chromosome separation". Science 297: 559-565) Kim Nasmyth noted that sister chromatid disengagement "occurs in the complete absence of microtubules and yet is capable of separating sister sequences by up to 0.5 µm." And he further remarks that this first phase in eukaryotic DNA segregation may involve processes similar to bacterial nucleoid segregation.
This comparison was made earlier (see Woldringh, C.L., and R. Van Driel. 1999. The Eukaryotic Perspective: Similarities and distinctions between pro- and eukaryotes. p. 77-90. In R.L. Charlebois (ed.), Organization of the prokaryotic genome. Chapter 5. American Society for Microbiology, Washington, D.C.). In this chapter we emphasized that the high amount of transcription occurring across the eukaryotic (mammalian) chromosome (see, for instance: The ENCODE Project Consortium. 2007. Nature 447: 799-816.) could play a role in strand separation just as has been suggested for bacterial segregation (see post by Samantha Orchard, May 14, 2007).
For me the question is how within the cell DNA sister-strands interact when a replication bubble is formed. If they separate under the influence of entropic forces as suggested by Jun and Mulder (2006. PNAS 103: 12388-12393), how long can this de-mixing last? Considered as a physical polymer probably not long. But as a "biological polymer", where the duplicated strands become decorated with DNA binding proteins with different and transient activities like ligation, transcription or supercoil formation, the initial de-mixing could become prolonged and stabilized, generating segregation.
Mentioning these processes reminds me of the remarks by Franklin Harold (his post of May 28, 2007) about spatial or self-organization arising from the interactions among large numbers of gene products. This is "statistical mechanics"! It is not the easiest physics for a biologist (see, for instance: Odijk, T. 1998. Osmotic compaction of supercoiled DNA into a bacterial nucleoid. Biophys. Chem. 73: 23-30). To get a feeling for this self-organization of macromolecules in either eukaryotes or prokaryotes we have to learn statistical thermodynamics and need time and patience to understand its old and difficult concepts, that can still hardly be applied to the complexity of cells.

[apalazzo]

I would say that a proper study of mitosis requires knowledge of the cytoskeleton and the ability to image the subject in question (you might even argue that the advent of imaging is cruitial for the understanding of cellular organization). Imaging and the study of the cytoskeleton are both easier in euks in comparison to proks.

[Paul Orwin]

This is a very interesting topic, and I have no explanation for why it has lagged eukaryotic studies. Perhaps because microbial genetic techniques are not well suited to studying partition defects? Hard to say if that is true, but molecular and bioinformatic strategies seem solid. It could a combination of bias (bacteria split simply, by fission) and difficulty (we never get chromosome partition mutants in screens, or we mis s the phenotype). Speaking of new studies on this topic, here's one that is in JBact ahead of print right now

Yoshiharu Yamaichi, Michael A. Fogel, Sarah M. McLeod, and Monica P. Hui
Distinct centromere-like parS sites on the two chromosomes of vibrio species
(sorry for the lack of formatting)

I just skimmed it, but it looks like they used the known ParAB gene function to identify the chromosomal sites required for correct partition of both chromosomes. As you said, a hot topic.

[Samantha Orchard]

I think it is interesting that while quite a bit is now known about the segregation of some bacterial plasmids (as described in this blog entry), relatively little is known about bacterial chromosome segregation. I wonder if it isn't a combination of the small size of the bacteria (as noted above) and the fact that any proteins required for chromosome segregation are essential for cell viability and are therefore less easily studied? Also, it should be noted that a few labs have proposed that bacterial chromosome segregation might occur without the need for specific segregation proteins. For example, see the work of Suckjoon Jun and Bela Mulder who propose a role for entropy in chromosome segregation (http://www.pnas.org/cgi/content/full/103/33/12388 ). Conrad Woldringh is another person I know of who is a proponent of a less active method of chromosome segregation, one involving transertion (combined transcription, translation and insertion of the proteins being translated into the membrane causing a "tug" on the chromosome) and diffusion of the daughter chromosomes due to phase exclusion from the solutes in the cytoplasm.

Elio replies:
Many thanks for pointing out that currently there is interest in a purely physical model for bacterial chromosome segregation, one not involving a special machinery. Surely this will be a hot area of debate. Stay tuned.