A Model for DNA Seg­re­ga­tion in 1963

Pic­tures Con­sid­ered #32

by Nanne Nan­ninga

Fig­ure 1. A Two cir­cu­lar repli­cons, the bac­te­r­ial chro­mo­some (Chr.) and a plas­mid (F) are at­tached to "the equa­to­r­ial perime­ter", which re­presents the "unit of seg­re­ga­tion". B The "cell sur­face is as­sumed to trans­mit to the repli­cons the sig­nal ini­ti­at­ing their repli­ca­tion". In C, D, and E "El­e­ments of the bac­te­r­ial mem­brane are as­sumed to grow be­tween the two planes of at­tach­ment of the daugh­ter repli­cons putting them pro­gres­sively apart". (I have short­ened the orig­i­nal leg­end, and el­e­ments of the text of the pa­per of Ja­cob et al. (1963) are be­tween quo­ta­tion marks). Source

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).

Fig­ure 2. Adapted ver­sion of Fig. 1. The first three items are men­tioned in the ac­com­pa­ny­ing text.

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).

  1. The bac­te­r­ial chro­mo­some (repli­con) is cir­cu­lar and at­tached to the equa­to­r­ial perime­ter (mem­brane).
  2. Its repli­ca­tion ma­chin­ery is like­wise at­tached to the mem­brane. At this site the chro­mo­some be­comes fully repli­cated, pro­duc­ing two repli­cons.
  3. Zonal en­ve­lope growth moves the sis­ter repli­cons apart.
  4. At the on­set of di­vi­sion the bac­te­r­ial chro­mo­somes have ac­quired their new cel­lu­lar po­si­tions.
  5. 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.

Fig­ure 3. DNA seg­re­ga­tion model of Ding­man (1974). Repli­ca­tion oc­curs in a fixed cel­lu­lar lo­ca­tion. Repli­cated DNA is threaded through the repli­ca­tion fac­tory as in­di­cated. Source

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. 

 

Ref­er­ences

  • Autissier F, and Kepes A (1971). Seg­re­ga­tion of mem­brane mark­ers dur­ing cell di­vi­sion in Es­cherichia coli. II. Seg­re­ga­tion of Lac-per­me­ase and Mel-per­me­ase stud­ied with a peni­cillin tech­nique. Biochim Bio­phys Acta, 249, 611–615.
  • Ca­de­nas E, and Gar­land PB (1979). Syn­the­sis of cy­to­plas­mic mem­brane dur­ing growth and di­vi­sion of Es­cherichia coli. Dis­per­sive be­hav­iour of res­pi­ra­tory ni­trate re­duc­tase. Biochem J, 184, 45–50.
  • Cole RM, and Hahn JJ (1962). Cell wall repli­ca­tion in Strep­to­coc­cus pyo­genes: im­muno­flu­o­res­cent meth­ods ap­plied dur­ing growth show that wall is formed equa­to­ri­ally. Sci­ence, 135, 722–724.
  • Crooke E. (2001). Es­cherichia coli DnaA protein–phospholipid in­ter­ac­tions: in vitro and in vivo. Biochimie, 83, 19–23.
  • Ding­man, CW (1974). Bidi­rec­tional chro­mo­some repli­ca­tion: some topo­log­i­cal con­sid­er­a­tions. J Theor Biol, 43, 187–195.
  • Gor­don GS, Sit­nikov D, Webb CD, Tele­man A, Straight A, Losick R, Mur­ray AW, and Wright A (1997). Chro­mo­some and low copy plas­mid seg­re­ga­tion in E. coli: vi­sual ev­i­dence for dis­tinct mech­a­nisms. Cell, 90, 1113–1121.
  • Green EW, and Schaechter M (1972). The mode of seg­re­ga­tion of the bac­te­r­ial cell mem­brane. Proc Natl Acad Sci USA, 69, 2312–2316.
  • Ja­cob F, Bren­ner S, and Cuzin F. (1963). On the reg­u­la­tion of DNA repli­ca­tion in bac­te­ria. Cold Spring Harb Symp Quant Biol, 28, 329–348.
  • Lin ECC, Hi­rota Y, and Ja­cob F (1971). On the process of cel­lu­lar di­vi­sion in Es­cherichia coli. J Bac­te­riol, 108, 375–385.
  • Mas­ters M, and Broda P (1971). Ev­i­dence for the bidi­rec­tional repli­ca­tions of the Es­cherichia coli chro­mo­some. Na­ture New Biol, 232, 137–140.
  • Niki H, and Hi­raga S (1998). Po­lar lo­cal­iza­tion of the repli­ca­tion ori­gin and ter­mi­nus in Es­cherichia coli nu­cleoids dur­ing chro­mo­some par­ti­tion­ing. Genes Dev, 12, 1036–1045.
  • Regev T, My­ers N, Zaraivach R, and Fishov I (2012). As­so­ci­a­tion of the chro­mo­some repli­ca­tion ini­tia­tor DnaA with the Es­cherichia coli in­ner mem­brane in vivo: quan­tity and mode of bind­ing. PLoS ONE, 7(5): e36441. doi: 10.1371/journal.pone.0036441.
  • Roos M, van Geel ABM, Aars­man MEG, Veuskens JTM, Woldringh CL, and Nan­ninga N (1999). Cel­lu­lar lo­cal­iza­tion of oriC dur­ing the cell cy­cle of Es­cherichia coli as an­a­lyzed by flu­o­res­cent in situ hy­bridiza­tion. Biochimie, 81, 797–802.
  • Trem­blay GY, Daniels MJ, and Schaechter M (1969). Iso­la­tion of a cell-mem­brane-DNA-nascent RNA com­plex from bac­te­ria. J Mol Biol, 40, 65–76.
  • Ver­wer R, and Nan­ninga N (1980). Pat­tern of meso-DL‑2,6‑diaminopimelic acid in­cor­po­ra­tion dur­ing the di­vi­sion cy­cle of Es­cherichia coli. J Bac­te­riol, 144, 27–336.
  • van Helvoort JM, and Woldringh, CL (1994). Nu­cleoid par­ti­tion­ing in Es­cherichia coli dur­ing steady-state growth and upon re­cov­ery from chlo­ram­pheni­col treat­ment. Mol Mi­cro­biol, 13,577–583.
  • Woldringh CL (1974). Mor­pho­log­i­cal analy­sis of nu­clear sep­a­ra­tion and cell di­vi­sion dur­ing the life cy­cle of Es­cherichia coli. J Bac­te­riol, 125, 248–257.

 

Nanne Nanninga

Nanne Nan­ninga is Emer­i­tus Pro­fes­sor of Mol­e­c­u­lar Cy­tol­ogy at the Uni­ver­sity of Am­s­ter­dam Swammer­dam In­sti­tute for Life Sci­ences

 

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