Tải bản đầy đủ - 0 (trang)
6 Two Novel Classes of Segregation Complex: Tubulin and Coiled-Coil Partition Factors

6 Two Novel Classes of Segregation Complex: Tubulin and Coiled-Coil Partition Factors

Tải bản đầy đủ - 0trang


F. Hayes and D. Barillà

depolymerizing spontaneously and rapidly in  vitro. It has been proposed that the

termini of TubZ/RepX polymers possess GTP caps that stabilize the GDP-bound

species within the filaments which are otherwise prone to disassembly (Chen and

Erickson 2008), akin to tubulin dynamics (Nogales and Wang 2006). The RepR

protein negatively regulates intracellular TubZ levels (Larsen et al. 2007). Although

the molecular basis for this regulation has yet to be clarified, RepR is a putative

DNA binding protein and may autoregulate the tubR-tubZ cassette at the transcriptional level. RepX also binds DNA, albeit weakly and non-specifically (Anand et al.

2008). It is unclear whether this activity reflects the protein’s role in plasmid segregation or replication: RepX is a probable replication initiation factor, as well as a

plasmid partitioning protein (Tinsley and Khan 2006). The mechanism by which

type III complexes segregate DNA remains to be determined, but it seems likely that

the polymerizing activity of TubZ/RepX plays a crucial role in plasmid movement.

Type IV partition modules are exemplified by the par gene of staphylococcal

plasmid pSK1. Homologous genes are located on plasmids in a diversity of Grampositive species (Simpson et al. 2003). The par gene is unusual as, by contrast with

type I–III systems, it appears to be the only gene necessary for plasmid stabilization

(Fig. 4.1). The Par protein is predicted to bind DNA via an N-terminal helix-turnhelix motif, with a possible binding site(s) in the region 5¢ of par that is rich in

repeat motifs and may act as a regulatory and/or centromeric sequence. A region of

coiled-coil structure is predicted in the C-terminal region of the protein (Simpson

et al. 2003). Further work is required to dissect the mode of plasmid stability mediated by type IV loci.

4.7 Perspectives

The plasmid segrosome provides a highly tractable framework to decipher the basis

of bacterial DNA segregation. Although plasmid partition cassettes most commonly are of type I, the molecular mechanisms that drive the segregation of plasmids bearing these modules still remain to be fully solved. Nevertheless, it has been

recently established that ParA homologues polymerize in response to nucleotide

binding in vitro, assemble into filamentous cytoskeletal structures in vivo, and traffic plasmids to specific subcellular locations within the cell. What is the purpose of

ParA polymerization during segregation? Like the actin-type ParM protein of type

II cassettes, bipolar polymerization of motor-like ParA proteins may propel plasmids attached to the fibre tips in opposite directions. Alternatively, bidirectional

disassembly of elongated ParA polymers may pull plasmids in opposite poleward


There is now convincing evidence that CBFs encoded by type I cassettes load on

to their centromeres and target plasmids to the mid-cell position where plasmid

pairing occurs mediated by these pre-segrosomal structures. Is there an intracellular

landmark to which the pre-segrosome is tethered at the cell centre? Data are also

accumulating that pre-segrosomes are nucleoprotein superstructures in which the

4  Extrachromosomal Components of the Nucleoid


CBF and centromeric DNA are arranged into intricate configurations. Considering

the diversity in plasmid centromere organization (Fig. 4.3) and in the sequences of

the CBFs that recognize them, it appears that pre-segrosomes assembled on different

plasmids may possess substantially different architectures. The interface of ParA

proteins with their cognate pre-segrosomes also may be specific. However, it is

plausible that the subsequent actions of homologous ParA factors during segregation are broadly similar.

In the case of type II partition cassettes, the ParR CBF apparently does not modulate filamentation of the actin-like ParM protein. By contrast, CBFs encoded by type

I modules not alone load on their centromeres, but also promote ParA protein polymerization. It is tempting to speculate that CBFs encoded by type I partition cassettes

are related functionally to the plethora of protein factors that regulate eukaryote

cytoskeletal dynamics. Moreover, the dual stimulation of ParA polymerization and

of ParA ATPase activity by these CBFs must be integrated during plasmid segregation

to ensure correct progression of the ParA polymerization-depolymerization cycle.

Furthermore, whereas the type II segrosome apparently can assemble and disassemble repeatedly during the bacterial cell cycle, it is unknown whether the same

pertains for type I complexes, or whether a single partitioning event is necessary and

sufficient for accurate plasmid maintenance. Further characterization of prototypical

segregation complexes, as well as of informative variant systems, will continue to

provide key mechanistic insights into genome segregation, a basic cellular process.

Acknowledgements  We apologize to authors whose valuable work could not be discussed in detail

or cited due to space limitations. Work in the authors’ laboratories is supported by the Biotechnology

and Biological Sciences Research Council (grants BB/G003114/1 and BB/F012004/1 to FH and DB,

respectively); the Medical Research Council (grant G0500588 to FH and DB, and grant G0801162

to DB); and, by European Union contract LSHM-CT-2005–019023 to FH.


Adachi S, Hori K, Hiraga S (2006) Subcellular positioning of F plasmid mediated by dynamic

localization of SopA and SopB. J Mol Biol 356:850–863

Ahmadian MR, Stege P, Scheffzek K, Wittinghofer A (1997) Confirmation of the arginine-finger

hypothesis for the GAP-stimulated GTP-hydrolysis reaction of Ras. Nat Struct Biol 4:686–689

Akhtar P, Anand SP, Watkins SC, Khan SA (2009) The tubulin-like RepX protein encoded by the

pXO1 plasmid forms polymers in vivo in Bacillus anthracis. J Bacteriol 191:2493–2500

Anand SP, Akhtar P, Tinsley E, Watkins SC, Khan SA (2008) GTP-dependent polymerization of

the tubulin-like RepX replication protein encoded by the pXO1 plasmid of Bacillus anthracis.

Mol Microbiol 67:881–890

Barillà D, Hayes F (2003) Architecture of the ParF-ParG protein complex involved in procaryotic

DNA segregation. Mol Microbiol 49:487–499

Barillà D, Rosenberg MF, Nobbmann U, Hayes F (2005) Bacterial DNA segregation dynamics

mediated by the polymerizing protein ParF. EMBO J 24:1453–1464

Barillà D, Carmelo E, Hayes F (2007) The tail of the ParG DNA segregation protein remodels

ParF polymers and enhances ATP hydrolysis via an arginine finger-like motif. Proc Natl Acad

Sci USA 104:1811–1816


F. Hayes and D. Barillà

Bartosik AA, Jagura-Burdzy G (2005) Bacterial chromosome segregation. Acta Biochim Pol


Becker E, Herrera NC, Gunderson FQ, Derman AI, Dance AL, Sims J, Larsen RA, Pogliano J

(2006) DNA segregation by the bacterial actin AlfA during Bacillus subtilis growth and development. EMBO J 25:5919–5931

Bork P, Sander C, Valencia A (1992) An ATPase domain common to prokaryotic cell cycle proteins,

sugar kinases, actin, and hsp70 heat shock proteins. Proc Natl Acad Sci USA 89:7290–7294

Bos JL, Rehmann H, Wittinghofer A (2007) GEFs and GAPs: critical elements in the control of

small G proteins. Cell 129:865–877

Bouet JY, Funnell BE (1999) P1 ParA interacts with the P1 partition complex at parS and an ATPADP switch controls ParA activities. EMBO J 18:1415–1424

Bouet JY, Ah-Seng Y, Benmeradi N, Lane D (2007) Polymerization of SopA partition ATPase:

regulation by DNA binding and SopB. Mol Microbiol 63:468–481

Breüner A, Jensen RB, Dam M, Pedersen S, Gerdes K (1996) The centromere-like parC locus of

plasmid R1. Mol Microbiol 20:581–592

Campbell CS, Mullins RD (2007) In vivo visualization of type II plasmid segregation: bacterial

actin filaments pushing plasmids. J Cell Biol 179:1059–1066

Carmelo E, Barillà D, Golovanov AP, Lian LY, Derome A, Hayes F (2005) The unstructured

N-terminal tail of ParG modulates assembly of a quaternary nucleoprotein complex in transcription repression. J Biol Chem 280:28683–28691

Castaing JP, Bouet JY, Lane D (2008) F plasmid partition depends on interaction of SopA with

non-specific DNA. Mol Microbiol 70:1000–1011

Chattoraj DK (2000) Control of plasmid DNA replication by iterons: no longer paradoxical. Mol

Microbiol 37:467–476

Chen Y, Erickson HP (2008) In vitro assembly studies of FtsZ/tubulin-like proteins (TubZ) from

Bacillus plasmids: evidence for a capping mechanism. J Biol Chem 283:8102–8109

Cooper JA, Sept D (2008) New insights into mechanism and regulation of actin capping protein.

Int Rev Cell Mol Biol 267:183–206

Dabrazhynetskaya A, Sergueev K, Austin S (2005) Species and incompatibility determination

within the P1par family of plasmid partition elements. J Bacteriol 187:5977–5983

Dabrazhynetskaya A, Brendler T, Ji X, Austin S (2009) Switching protein-DNA recognition

specificity by single amino acid substitutions in the P1 par family of plasmid partition elements. J Bacteriol 191:1126–1131

Dam M, Gerdes K (1994) Partitioning of plasmid R1. Ten direct repeats flanking the parA promoter constitute a centromere-like partition site parC, that expresses incompatibility. J Mol

Biol 236:1289–1298

Davey MJ, Funnell BE (1994) The P1 plasmid partition protein ParA. A role for ATP in sitespecific DNA binding. J Biol Chem 269:29908–29913

Davis MA, Martin KA, Austin SJ (1992) Biochemical activities of the ParA partition protein of

the P1 plasmid. Mol Microbiol 6:1141–1147

Davis MA, Radnedge L, Martin KA, Hayes F, Youngren B, Austin SJ (1996) The P1 ParA protein

and its ATPase activity play a direct role in the segregation of plasmid copies to daughter cells.

Mol Microbiol 21:1029–1036

de la Hoz AB, Ayora S, Sitkiewicz I, Fernández S, Pankiewicz R, Alonso JC, Ceglowski P (2000)

Plasmid copy-number control and better-than-random segregation genes of pSM19035 share a

common regulator. Proc Natl Acad Sci USA 97:728–733

de la Hoz AB, Pratto F, Misselwitz R, Speck C, Weihofen W, Welfle K, Saenger W, Welfle H,

Alonso JC (2004) Recognition of DNA by w protein from the broad-host range Streptococcus

pyogenes plasmid pSM19035: analysis of binding to operator DNA with one to four heptad

repeats. Nucleic Acids Res 32:3136–3147

del Solar G, Espinosa M (2000) Plasmid copy number control: an ever-growing story. Mol

Microbiol 37:492–500

Delbrück H, Ziegelin G, Lanka E, Heinemann U (2002) An Src homology 3-like domain is

responsible for dimerization of the repressor protein KorB encoded by the promiscuous IncP

plasmid RP4. J Biol Chem 277:4191–4198

4  Extrachromosomal Components of the Nucleoid


Derman AI, Lim-Fong G, Pogliano J (2008) Intracellular mobility of plasmid DNA is limited by

the ParA family of partitioning systems. Mol Microbiol 67:935–946

Derome A, Hoischen C, Bussiek M, Grady R, Adamczyk M, Kędzierska B, Diekmann S, Barillà

D, Hayes F (2008) Centromere anatomy in the multidrug-resistant pathogen Enterococcus

faecium. Proc Natl Acad Sci USA 105:2151–2156

Dmowski M, Sitkiewicz I, Ceglowski P (2006) Characterization of a novel partition system encoded

by the d and w genes from the streptococcal plasmid pSM19035. J Bacteriol 188:4362–4372

Ebersbach G, Gerdes K (2001) The double par locus of virulence factor pB171: DNA segregation

is correlated with oscillation of ParA. Proc Natl Acad Sci USA 98:15078–15083

Ebersbach G, Gerdes K (2005a) Plasmid segregation mechanisms. Annu Rev Genet 39:453–479

Ebersbach G, Gerdes K (2005b) Bacterial mitosis: partitioning protein ParA oscillates in spiralshaped structures and positions plasmids at mid-cell. Mol Microbiol 52:385–398

Ebersbach G, Ringgaard S, Moller-Jensen J, Wang Q, Sherratt DJ, Gerdes K (2006) Regular cellular distribution of plasmids by oscillating and filament-forming ParA ATPase of plasmid

pB171. Mol Microbiol 61:1428–1442

Edgar R, Chattoraj DK, Yarmolinsky M (2001) Pairing of P1 plasmid partition sites by ParB. Mol

Microbiol 42:1363–1370

Fogel MA, Waldor MK (2006) A dynamic, mitotic-like mechanism for bacterial chromosome

segregation. Genes Dev 20:3269–3282

Fothergill TJG, Barillà D, Hayes F (2005) Protein diversity confers specificity in plasmid segregation. J Bacteriol 187:2651–2661

Friedman SA, Austin SJ (1988) The P1 plasmid-partition system synthesizes two essential proteins from an autoregulated operon. Plasmid 19:103–112

Fung E, Bouet JY, Funnell BE (2001) Probing the ATP-binding site of P1 ParA: partition and repression have different requirements for ATP binding and hydrolysis. EMBO J 20:4901–4911

Funnell BE (1988) Participation of Escherichia coli integration host factor in the P1 plasmid partition system. Proc Natl Acad Sci USA 85:6657–6661

Funnell BE (1991) The P1 plasmid partition complex at parS. The influence of Escherichia coli

integration host factor and of substrate topology. J Biol Chem 266:14328–14337

Funnell BE (2005) Partition-mediated plasmid pairing. Plasmid 53:119–125

Garner EC, Campbell CS, Mullins RD (2004) Dynamic instability in a DNA-segregating prokaryotic actin homolog. Science 306:1021–1025

Garner EC, Campbell CS, Weibel DB, Mullins RD (2007) Reconstitution of DNA segregation

driven by assembly of a prokaryotic actin homolog. Science 315:1270–1274

Golovanov AP, Barillà D, Golovanova M, Hayes F, Lian LY (2003) ParG, a protein required for

active partition of bacterial plasmids, has a dimeric ribbon-helix-helix structure. Mol Microbiol


Hatano T, Yamaichi Y, Niki H (2007) Oscillating focus of SopA associated with filamentous

structure guides partitioning of F plasmid. Mol Microbiol 64:1198–1213

Hayes F (2000) The partition system of multidrug resistance plasmid TP228 includes a novel

protein that epitomizes an evolutionarily-distinct subgroup of the ParA superfamily. Mol

Microbiol 37:528–541

Hayes F (2003a) The function and organization of plasmids. Methods Mol Biol 235:1–17

Hayes F (2003b) Toxins-antitoxins: plasmid maintenance, programmed cell death, and cell cycle

arrest. Science 301:1496–1499

Hayes F, Austin SJ (1993) Specificity determinants of the P1 and P7 plasmid centromere analogs.

Proc Natl Acad Sci USA 90:9228–9232

Hayes F, Austin S (1994) Topological scanning of the P1 plasmid partition site. J Mol Biol 243:190–198

Hayes F, Barillà D (2006a) The bacterial segrosome: a dynamic nucleoprotein machine for DNA

trafficking and segregation. Nat Rev Microbiol 4:133–143

Hayes F, Barillà D (2006b) Assembling the bacterial segrosome. Trends Biochem Sci 31:247–250

Hayes F, Davis MA, Austin SJ (1993) Fine-structure analysis of the P7 plasmid partition site.

J Bacteriol 175:3443–3451

Hayes F, Radnedge L, Davis MA, Austin SJ (1994) The homologous operons for P1 and P7 plasmid

partition are autoregulated from dissimilar operator sites. Mol Microbiol 11:249–260


F. Hayes and D. Barillà

Hoischen C, Bolshoy A, Gerdes K, Diekmann S (2004) Centromere parC of plasmid R1 is curved.

Nucleic Acids Res 32:5907–5915

Hoischen C, Bussiek M, Langowski J, Diekmann S (2008) Escherichia coli low-copy-number

plasmid R1 centromere parC forms a U-shaped complex with its binding protein ParR.

Nucleic Acids Res 36:607–615

Jensen RB, Gerdes K (1997) Partitioning of plasmid R1. The ParM protein exhibits ATPase activity and interacts with the centromere-like ParR-parC complex. J Mol Biol 269:505–513

Jensen RB, Dam M, Gerdes K (1994) Partitioning of plasmid R1. The parA operon is autoregulated by ParR and its transcription is highly stimulated by a downstream activating element.

J Mol Biol 236:1299–1309

Jensen RB, Lurz R, Gerdes K (1998) Mechanism of DNA segregation in prokaryotes: replicon

pairing by parC of plasmid R1. Proc Natl Acad Sci USA 95:8550–8555

Khare D, Ziegelin G, Lanka E, Heinemann U (2004) Sequence-specific DNA binding determined

by contacts outside the helix-turn-helix motif of the ParB homolog KorB. Nat Struct Mol Biol


Koonin EV (1993) A superfamily of ATPases with diverse functions containing either classical or

deviant ATP-binding motif. J Mol Biol 229:1165–1174

Larsen RA, Cusumano C, Fujioka A, Lim-Fong G, Patterson P, Pogliano J (2007) Treadmilling of

a prokaryotic tubulin-like protein, TubZ, required for plasmid stability in Bacillus thuringiensis.

Genes Dev 21:1340–1352

Leonard TA, Butler PJ, Lowe J (2005) Bacterial chromosome segregation: structure and DNA

binding of the Soj dimer – a conserved biological switch. EMBO J 24:270–282

Li Y, Austin S (2002) The P1 plasmid is segregated to daughter cells by a ‘capture and ejection’

mechanism coordinated with Escherichia coli cell division. Mol Microbiol 46:63–74

Li Y, Dabrazhynetskaya A, Youngren B, Austin S (2004) The role of Par proteins in the active

segregation of the P1 plasmid. Mol Microbiol 53:93–102

Libante V, Thion L, Lane D (2001) Role of the ATP-binding site of SopA protein in partition of

the F plasmid. J Mol Biol 314:387–399

Lim GE, Derman AI, Pogliano J (2005) Bacterial DNA segregation by dynamic SopA polymers.

Proc Natl Acad Sci USA 102:17658–17663

Lowe J, Amos LA (2009) Evolution of cytomotive filaments: the cytoskeleton from prokaryotes

to eukaryotes. Int J Biochem Cell Biol 41:323–329

Lynch AS, Wang JC (1995) SopB protein-mediated silencing of genes linked to the sopC locus of

Escherichia coli F plasmid. Proc Natl Acad Sci USA 92:1896–1900

Machón C, Fothergill TJG, Barillà D, Hayes F (2007) Promiscuous stimulation of ParF protein

polymerization by heterogeneous centromere binding factors. J Mol Biol 374:1–8

Marston AL, Errington J (1999) Dynamic movement of the ParA-like Soj protein of B. subtilis

and its dual role in nucleoid organization and developmental regulation. Mol Cell 4:673–682

Moller-Jensen J, Jensen RB, Lowe J, Gerdes K (2002) Prokaryotic DNA segregation by an actinlike filament. EMBO J 21:3119–3127

Moller-Jensen J, Borch J, Dam M, Jensen RB, Roepstorff P, Gerdes K (2003) Bacterial mitosis:

ParM of plasmid R1 moves plasmid DNA by an actin-like insertional polymerization mechanism. Mol Cell 12:1477–1487

Moller-Jensen J, Ringgard S, Mercogliano CP, Gerdes K, Lowe J (2007) Structural analysis of the

ParR/parC plasmid partition complex. EMBO J 26:4413–4422

Motallebi-Veshareh M, Rouch DA, Thomas CM (1990) A family of ATPases involved in active

partitioning of diverse bacterial plasmids. Mol Microbiol 4:1455–1463

Murayama K, Orth P, de la Hoz AB, Alonso JC, Saenger W (2001) Crystal structure of w transcriptional repressor encoded by Streptococcus pyogenes plasmid pSM19035 at 1.5 Å resolution.

J Mol Biol 314:789–796

Nadanaciva S, Weber J, Wilke-Mounts S, Senior AE (1999) Importance of F1-ATPase residue

a-Arg-376 for catalytic transition state stabilization. Biochemistry 38:15493–15499

Nogales E, Wang HW (2006) Structural mechanisms underlying nucleotide-dependent selfassembly of tubulin and its relatives. Curr Opin Struct Biol 16:221–229

4  Extrachromosomal Components of the Nucleoid


Nordstrom K (2006) Plasmid R1 – replication and its control. Plasmid 55:1–26

Orlova A, Garner EC, Galkin VE, Heuser J, Mullins RD, Egelman EH (2007) The structure of

bacterial ParM filaments. Nat Struct Mol Biol 14:921–926

Pogliano J (2008) The bacterial cytoskeleton. Curr Opin Cell Biol 20:19–27

Popp D, Yamamoto A, Iwasa M, Narita A, Maeda K, Maéda Y (2007) Concerning the dynamic

instability of actin homolog ParM. Biochem Biophys Res Commun 353:109–114

Popp D, Narita A, Oda T, Fujisawa T, Matsuo H, Nitanai Y, Iwasa M, Maeda K, Onishi H, Maéda

Y (2008) Molecular structure of the ParM polymer and the mechanism leading to its nucleotidedriven dynamic instability. EMBO J 27:570–579

Pratto F, Cicek A, Weihofen WA, Lurz R, Saenger W, Alonso JC (2008) Streptococcus pyogenes

pSM19035 requires dynamic assembly of ATP-bound ParA and ParB on parS DNA during

plasmid segregation. Nucleic Acids Res 36:3676–3689

Quisel JD, Lin DC, Grossman AD (1999) Control of development by altered localization of a

transcription factor in B. subtilis. Mol Cell 4:665–672

Radnedge L, Davis MA, Austin SJ (1996) P1 and P7 plasmid partition: ParB protein bound to its

partition site makes a separate discriminator contact with the DNA that determines species

specificity. EMBO J 15:1155–1162

Radnedge L, Youngren B, Davis M, Austin S (1998) Probing the structure of complex macromolecular interactions by homolog specificity scanning: the P1 and P7 plasmid partition systems.

EMBO J 17:6076–6085

Ringaard S, Ebersbach G, Borch J, Gerdes K (2007a) Regulatory cross-talk in the double par

locus of plasmid pB171. J Biol Chem 282:3134–3145

Ringaard S, Lowe J, Gerdes K (2007b) Centromere pairing by a plasmid-encoded type I ParB

protein. J Biol Chem 282:28216–28225

Rodionov O, Yarmolinsky M (2004) Plasmid partitioning and the spreading of P1 partition protein

ParB. Mol Microbiol 52:1215–1223

Rodionov O, Lobocka M, Yarmolinsky M (1999) Silencing of genes flanking the P1 plasmid

centromere. Science 283:546–549

Rothfield L, Taghbalout A, Shih YL (2005) Spatial control of bacterial division-site placement.

Nat Rev Microbiol 3:959–968

Salje J, Lowe J (2008) Bacterial actin: architecture of the ParMRC plasmid DNA partitioning

complex. EMBO J 27:2230–2238

Salje J, Zuber B, Lowe J (2009) Electron cryomicroscopy of E. coli reveals filament bundles

involved in plasmid DNA segregation. Science 323:509–512

Schumacher MA (2007) Structural biology of plasmid segregation proteins. Curr Opin Struct Biol


Schumacher MA (2008) Structural biology of plasmid partition: uncovering the molecular mechanisms of DNA segregation. Biochem J 412:1–18

Schumacher MA, Funnell BE (2005) Structures of ParB bound to DNA reveal mechanism of partition

complex formation. Nature 438:516–519

Schumacher MA, Glover TC, Brzoska AJ, Jensen SO, Dunham TD, Skurray RA, Firth N (2007a)

Segrosome structure revealed by a complex of ParR with centromere DNA. Nature 450:


Schumacher MA, Mansoor A, Funnell BE (2007b) Structure of a four-way bridged ParB-DNA

complex provides insight into P1 segrosome assembly. J Biol Chem 282:10456–10464

Sergueev K, Dabrazhynetskaya A, Austin S (2005) Plasmid partition system of the P1par family

from the pWR100 virulence plasmid of Shigella flexneri. J Bacteriol 187:3369–3373

Simpson AE, Skurray RA, Firth N (2003) A single gene on the staphylococcal multiresistance

plasmid pSK1 encodes a novel partitioning system. J Bacteriol 185:2143–2152

Summers D (1998) Timing, self-control and a sense of direction are the secrets of multicopy

plasmid stability. Mol Microbiol 29:1137–1145

Thanbichler M, Shapiro L (2006) Chromosome organization and segregation in bacteria. J Struct

Biol 156:292–303

Thomas CM (ed) (2000) The horizontal gene pool. Harwood Academic, Amsterdam, The Netherlands


F. Hayes and D. Barillà

Tinsley E, Khan SA (2006) A novel FtsZ-like protein is involved in replication of the anthrax

toxin-encoding pXO1 plasmid in Bacillus anthracis. J Bacteriol 188:2829–2835

van den Ent F, Moller-Jensen J, Amos LA, Gerdes K, Lowe J (2002) F-actin-like filaments formed

by plasmid segregation protein ParM. EMBO J 21:6935–6943

Vecchiarelli AG, Schumacher MA, Funnell BE (2007) P1 partition complex assembly involves

several modes of protein-DNA recognition. J Biol Chem 282:10944–10952

Watanabe E, Wachi M, Yamasaki M, Nagai K (1992) ATPase activity of SopA, a protein essential

for active partitioning of F plasmid. Mol Gen Genet 234:346–352

Weihofen WA, Cicek A, Pratto F, Alonso JC, Saenger W (2006) Structures of w repressors bound

to direct and inverted DNA repeats explain modulation of transcription. Nucleic Acids Res


Zampini M, Derome A, Bailey SES, Barillà D, Hayes F (2009) Recruitment of the ParG segregation

protein to different affinity DNA sites. J Bacteriol 191:3832–3841

Chapter 5

Nucleoid Structure and Segregation

Conrad L. Woldringh

Abstract  One of the fundamental differences between eukaryotic and bacterial cell

cycles is the possibility of re-replication in bacteria. The organization of the bacterial

DNA in the nucleoid and the mechanism of its segregation make it possible that during

an ongoing round of replication, new initiations can take place. In contrast to the

protein-rich, nucleosomal structure of eukaryotic DNA, the bacterial chromosome is

thought to consist of thousands of supercoiled, branched segments that are compacted

in a protein-poor nucleoid phase through non-specific volume-exclusion interactions

with cytoplasmic proteins. Recent findings on the movement of fluorescent loci on

both arms (replichores) of the Escherichia coli chromosome and on the dynamics of

DNA polymerases have given a coherent picture of nucleoid organization. While the

chromosome is replicated bidirectionally from a single origin by independent replisomes, the daughter strands separate and move past the bulk of unreplicated DNA in

a simultaneous replication/segregation process. Repulsive, entropic forces probably

promote the segregation of DNA daughter strands. In E. coli the two replichores of

each separating daughter strand can move apart at different velocities and become

positioned in two halves of the newly synthesized nucleoid with the origin in between.

In contrast, in Caulobacter crescentus and Vibrio cholerae (oriCI), one origin is kept

near the old cell pole and both replichores move together with the other origin at the tip

of the newly synthesized nucleoid. More accurate determinations of the dynamics of

DNA loci under various growth conditions and during growth inhibition (run-off DNA

synthesis) may be expected to establish whether entropic forces are sufficient for DNA

segregation or whether dedicated biological mechanisms have to give an extra drift, for

instance in initial origin separation or in establishing the division of the nucleoid.

Keywords  Bacterial chromosome • chromosome arms (replichores) • entropic

repulsion of DNA strands • nucleoid segregation • phase separation • supercoiled

polymer-network • volume–exclusion interactions

C.L. Woldringh (*)

Molecular Cytology, Faculty of Science, Swammerdam Institute for Life Sciences,

University of Amsterdam, Amsterdam, The Netherlands

e-mail: c.l.woldringh@uva.nl

R.T. Dame and C.J. Dorman (eds.), Bacterial Chromatin,

DOI 10.1007/978-90-481-3473-1_5, © Springer Science+Business Media B.V. 2010



C.L. Woldringh

5.1 Introduction

In bacteria chromosomes usually contain a single origin of replication from which

two replisomes start to replicate the chromosome arms in opposite directions

(Fig. 5.1a). Fast growing E. coli cells (Fig. 5.1b) divide more quickly than they can

produce a complete chromosome, as the replication time (C-period) may be longer

than the generation time (Td). To produce complete, viable cells, the cell has to

initiate DNA replication in step with cell growth and at the same frequency as it

divides, i.e. once per cell cycle (Helmstetter and Cooper 1968; Hansen et al. 1991).

During fast growth this implies that initiation occurs on a chromosome that is still

carrying out one or even two round(s) of replication. This so-called multi-fork replication during overlapping C-periods is only possible if newly replicated daughter

strands are separated as soon as they are synthesized, in a concurrent replication

and segregation process (right panels in Fig. 5.1a, b). This avoids the possibility of

entanglement of newly replicated strands and avoids the necessity for a disentangling mechanism that has to recognize the hierarchy of the replicated strands

(Donachie et al. 1995).

Eukaryotic cells have no mechanism for beginning a new DNA replication cycle

before finishing the last one. Such re-replications or overlapping S-phases are not

possible, but also not necessary because eukaryotes can start DNA replication at

many origins. Chromosome replication thus occurs in a period (S + G2/M) that is

always shorter than the cell’s generation time. In a first stage of segregation

(Fig.  5.1c), sister chromatids are aligned, held together by cohesins (Nasmyth

2002) and subsequently disentangled by a mechanism that may involve chromosome condensation by condensins (Marko and Siggia 1997); this mechanism is still

not well understood (Guacci 2007). In a second stage of segregation (Fig.  5.1d),

microtubules attach to a single, kinetochore built on the aligned centromeres of the

compact chromosomes. This mechanism, mitosis, is unsuitable for separating replicating chromosomes, let alone chromosomes that are in a state of more than one

round of replication like in E. coli (Fig. 5.1b).

The use of fluorescence microscopy to study bacterial strains with fluorescent

DNA markers has greatly advanced our understanding of bacterial chromosome

organization (Gordon et al. 1997; Niki et al. 2000; Wang et al. 2005; Nielsen et al.

2006a). However, the finding of rapid movements of fluorescently labeled origins

has led some authors to consider that bacterial chromosome segregation occurs by

a mechanism similar to that in eukaryotes. This also led them to adopt a eukaryotic

terminology for the bacterial cell cycle (e.g. Gitai et al. 2005; Fogel and Waldor

2006). This terminology, however, conceals a fundamental difference between the

two systems of DNA replication: the impossibility in eukaryotic cells of concurrent

DNA replication and segregation (i.e. overlapping S-phases) and the absence in

bacteria of a G2/M period in which chromosome condensation takes place. Thus,

as has been suggested earlier (Nasmyth 2002; Woldringh and van Driel 1999),

bacterial segregation can better be compared with the first stage of disentanglement

of the eukaryotic chromatids (Fig.  5.1c), than with the second stage, mitosis

5  Nucleoid Structure and Segregation


Fig. 5.1  Schematic representation of chromosome replication and segregation in pro- and eukaryotes.

Cell sizes are not to scale. Origins of replication are indicated by circles, replisomes by triangles.

(a) The circular chromosome of a bacterium like E. coli is replicated during the C-period (slow

growth conditions). Different replichores are in black and grey; different daughter strands are in full

or dashed lines. (b) During fast growth, re-replications from multiple origins occur every generation

time, Td, which is shorter than the C-period. Right panels in (a) and (b): simultaneous replication

and segregation of the origins. Note larger cell in B as predicted by the Helmstetter-Cooper model

(1968). Different daughter strands are in full and dashed lines. (c) The linear chromosome of a

eukaryotic cell like yeast is replicated from many origins. Different daughter strands (chromatids)

are in black and grey. The nucleosomal structure of the DNA is not indicated. The replisomes occur

in replication factories (grey circles). Replication is coupled with cohesion by cohesin complexes

indicated by ovals. Groups of replisome pairs can combine in a single factory (see Kitamura et al.

2006). (d) Sister chromatids are disentangled, aligned and condensed by condensin complexes in the

yeast nucleus. Final segregation occurs after microtubules have attached to the kinetochore structure

(black square) and cohesins have become proteolyzed

(Fig.  5.1d), in which the condensed chromatids are pulled to opposite cell poles

with the help of microtubules.

In this review I will first discuss cytological and physical aspects of the structure

of the bacterial nucleoid both in situ and in isolated nucleoids. In subsequent sections a rather coherent picture of segregation is given based on recent findings of

the dynamics of fluorescent DNA markers and replisomes. I will argue that replication


C.L. Woldringh

occurs in the outer border of the nucleoid where entropic, repulsive interactions

between newly replicated segments of the chromosome cause their segregation.

5.2 Nucleoid Structure

5.2.1 Conclusions Based on Light Microscopy, Electron

Microscopy and Cryoelectron Tomography

That bacterial DNA is found in visible structures called nucleoids is clear for fastgrowing, relatively large E. coli cells. This was demonstrated in 1956 in elegant

phase-contrast images by Mason and Powelson (1956) and have to my knowledge

only been reproduced with the same quality by Hironori Niki (National Institute of

Genetics, Japan, unpublished). These timelapse sequences of cells embedded in

gelatin indicate that the bacterial nucleoid is a low mass-density region that enlarges

and duplicates in step with cell growth and division. In smaller cells, like slowgrowing E. coli or Caulobacter crescentus, distinct nucleoids are difficult to see

using either phase contrast or fluorescence microscopy. Indeed Caulobacter cells

stained with DAPI (6-diamidino-2phenylindole dihydrochloride hydrate) were first

reported to have no chromosome-free regions and thus no nucleoids (Jensen and

Shapiro 1999; Jensen 2006). However, combined phase-contrast and fluorescence

microscopy suggests that there is a cytoplasmic phase along the cell border and that

the DNA is not fully dispersed throughout the cell (Fig. 5.2a, left panel). The phase

separation in the small cell is made more obvious by fixation with osmium tetroxide (Fig. 5.2a, right panel) and by electron microscopy of thin sections of both

C. crescentus (Poindexter 1964) and E. coli cells (Woldringh et  al. 1977). Such

electron micrographs show occasional fibrils which penetrate from the nucleoid

into the cytoplasm, possibly connecting even to the plasma membrane (van Iterson

1965). However, the phase separation in osmium-tetroxide fixed cells could be an

artifact because fixation with glutaraldehyde does not reveal such a clear separation

between a fibrillar nucleoid and a granular cytoplasm (see for reviews Woldringh

and Nanninga 1985; Robinow and Kellenberger 1994).

Does this mean that in small, live cells the DNA is dispersed throughout the

cytoplasm and that there is no separate cytoplasmic phase as seen in the large, live cells

of Mason and Powelson (1956)? It was hoped that this question could be answered

by the introduction of cryofixation techniques. However, in freeze-fracture preparations nucleoids were not always visible (Nanninga 1969). Also with the more

recently developed technique of cryoelectron tomography a nucleoid is not always

present, e.g. in tomograms of the very small bacterium Spiroplasma melliferum

(Fig. 5.2e; Ortiz et al. 2006). It is therefore relevant that ribosome-free regions with

a different texture could be seen in vitrified sections of Deinococcus radiodurans

(Eltsov and Dubochet 2005) and in cryoelectron tomograms of Bdellovibrio bacteriovorus (Fig.  5.2d; Borgnia et  al. 2008). The comparison of cell and nucleoid

sizes of these different bacteria (Fig. 5.2b–e) shows that nucleoid segregation in the

Tài liệu bạn tìm kiếm đã sẵn sàng tải về

6 Two Novel Classes of Segregation Complex: Tubulin and Coiled-Coil Partition Factors

Tải bản đầy đủ ngay(0 tr)