by Nanne Nanninga
The classic paper on 'The Regulation of DNA Replication in Bacteria' by Jacob et al. (1963) included a model on bacterial DNA segregation (Fig. 1). In retrospect the model had far reaching consequences which can still be felt today. It entailed a research program that inspired many scientists. Yet, the model was not the main topic of the paper. Actually, its focus was directed at the mechanism of bacterial DNA replication and in particular towards the development of the concept of the replicon. However, the question was also posed how replicated DNA could be distributed over new daughter cells. This was a pressing problem and the more so "after withdrawal of the concept of the occurrence of classical mitosis in bacteria" (Delamater, 1962). The model was an attempt to account for the absence of knowledge about bacterial DNA segregation and thus also an attempt to fill the void left by Delamater. To compensate for the lack of knowledge, French logic has been applied. That is to say, a link was hypothesized between cell elongation and membrane-attachment of replicated DNA: "The cell membrane playing the role of mitotic apparatus". And "the bacterial surface playing the role of both the centrosome and the centromere of cells of higher organisms" (Jacob et al., 1963).
For the sake of clarity I have taken the liberty to redraw the model (Fig. 2) while emphasizing the main points. (Note that I have left out the segregation of a replicated plasmid (F factor), which might warrant a separate treatment (see here in STC).
- The bacterial chromosome (replicon) is circular and attached to the equatorial perimeter (membrane).
- Its replication machinery is likewise attached to the membrane. At this site the chromosome becomes fully replicated, producing two replicons.
- Zonal envelope growth moves the sister replicons apart.
- At the onset of division the bacterial chromosomes have acquired their new cellular positions.
- This is followed by cell division.
With the exception of the circularity of the bacterial chromosome, in 1963 there was no scientific evidence for all other elements of the model, thus providing for a popular (in hindsight) and extensive research program all over the world (including myself). I will address the first three of the above items one by one and briefly comment on their past and/or present status.
1. In 1963 the circularity of the bacterial chromosome had already been inferred on the basis of genetics. As is well known, conjugation experiments employing E. coli Hfr strains with the F factor integrated at different chromosomal sites revealed that in E. coli "the genetic information is contained along a linear structure, the bacterial 'chromosome', which seems to exist as a closed ring" (Jabob et al., 1963, and references therein). Visually this was corroborated by the microscopic image of the replicating DNA molecule in 1963 (see Pictures Considered #2).
2a. DNA-membrane attachment as a topic witnessed a plethora of publications up till today. Subjects included the question whether DNA is at all attached and if so are the attachments specific for a chromosomal region. For example, the so-called M-band technique (Tremblay et al., 1969) has been widely used to analyse DNA-membrane attachment by cell fractionation. In the course of time, attention became directed to the DNA-binding protein DnaA, which is involved in the initiation of DNA replication. The membrane connection lies in the observation that acid phospholipids affect the activity of DnaA in E. coli in vitro (Crooke, 2001 and references therein). Whether this reflects a specific physiological requirement is not yet certain. The intricacies of the subject have been clearly outlined by Regev et al. (2012). Today, after more than 50 years since its inception, a definite answer on DNA-membrane attachment is not yet forthcoming.
2b. Also indicated in the figure is the complete replication of the bacterial chromosome before segregation starts. This is clearly derived from eukaryotic mitosis where chromosomes move apart after their duplication. Now we know that DNA replication is bidirectional (Masters and Broda, 1971) and that replication and segregation go hand in hand. Initially, the latter could be inferred from elecron microscopic images of thin sections of E. coli showing a correlation between cell length and the length of the nucleoid (Woldring, 1974). A breakthrough was the marking of E. coli oriC with fluorescent probes in fixed (Niki & Hiraga, 1998) or living cells (Gordon et al., 1997). Studies with fluorescently labeled origins in well-defined steady state grown cultures demonstrated their moving apart (Roos et al., 1999) in accordance with a centrally-located replication factory through which replicated DNA is threaded (Fig. 3; Dingman, 1974). Thus, as in the scheme of Jacob et al., replication takes place in the cell center, though in contrast, newly-replicated DNA does not stay there.
3. Can the cell membrane play the role of a mitotic apparatus? Inspiration derived clearly, as stated by Jacob et al., from the elegant work of Cole and Hahn (1962) on wall replication in the spherical Streptococcus pyogenes cells. Cole and Hahn showed alternating fluorescent and non-fluorescent zones in chains of S. pyogenes, reflecting old and new cell wall, respectively (see Pictures Considered #7). Does zonal envelope growth apply to a rod-shaped cell like E. coli as proposed by Jacob et al. ? Remember that in 1963 membrane fluidity as a concept was still non-existent. By contrast, the focus was on the stable unit membrane. Many attempts have been carried out to deduce the mode of membrane extension, being (semi)-conservative or dispersive. Experiments involved inducible membrane proteins or radioactive chemical membrane constituants. Early attempts pointed to a dispersive (Lin et al., 1971) or conservative (Autissier & Kepes, 1971) membrane growth. In subsequent experiments, the dispersive mode became the dominant point of view (Green & Schaechter, 1972; Cadenas & Garland, 1979). Consequently, a role for the cell membrane as part of a bacterial mitotic apparatus became unlikely. Because the cell membrane is part of the envelope which includes the covalently closed peptidoglycan layer, a mitotic role for the latter might be considered. However, also in this case zonal assembly could not be demonstrated (Verwer & Nanninga, 1980; Woldringh et al., 1987; de Pedro et al., 1997). Also regarding the E. coli outer membrane which is tightly attached to the peptidoglycan layer by lipoprotein dispersive insertion of the latter was observed (Hiemstra et al., 1987). Collectively, these findings suggest that bacterial chromosomal DNA segregation (which is not mitosis) proceeds independent from the cell envelope.
So, now that we are reasonably sure that we know how chromosome segregation does not take place in bacteria, all that's left is to find out how it does! It has been noted that the segregating nucleoid keeps a fixed distance to the cell poles (van Helvoort & Woldringh, 1994), implicating perhaps that oriC interacts with a pole through a hypothetical proteinaceous framework.
One might wonder why the influence of the model of Jacob et al. persisted for such a long time. Perhaps the answer lies in the fact that bacteria are so small, thus precluding the straightforward application of eukaryotic cellular organization.
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Nanne Nanninga is Emeritus Professor of Molecular Cytology at the University of Amsterdam Swammerdam Institute for Life Sciences