We have investigated the replication patterns of the two chromosomes of the bacterium Vibrio cholerae grown in four different media. By combining flow cytometry and quantitative real-time PCR with computer simulations, we show that in rich media, V. cholerae cells grow with overlapping replication cycles of both the large chromosome (ChrI) and the small chromosome (ChrII). In Luria–Bertani (LB) medium, initiation occurs at four copies of the ChrI origin and two copies of the ChrII origin. Replication of ChrII was found to occur at the end of the ChrI replication period in all four growth conditions. Novel cell-sorting experiments with marker frequency analysis support these conclusions. Incubation with protein synthesis inhibitors indicated that the potential for initiation of replication of ChrII was present at the same time as that of ChrI, but was actively delayed until much of ChrI was replicated. Investigations of the localization of SeqA bound to new DNA at replication forks indicated that the forks were co-localized in pairs when cells grew without overlapping replication cycles and in higher-order structures during more rapid growth. The increased degree of fork organization during rapid growth may be a means by which correct segregation of daughter molecules is facilitated.
Allardet-ServentA.,
Michaux-CharachonS.,
Jumas-BilakE.,
KarayanL.,
RamuzM.1993; Presence of one linear and one circular chromosome in the Agrobacterium tumefaciens C58 genome. J Bacteriol 175:7869–7874
BremerH.,
ChurchwardG.1977; An examination of the Cooper–Helmstetter theory of DNA replication in bacteria and its underlying assumptions. J Theor Biol 69:645–654
BrendlerT.,
AbelesA.,
AustinS.1995; A protein that binds to the P1 origin core and the oriC 13mer region in a methylation-specific fashion is the product of the host seqA gene. EMBO J 14:4083–4089
BrendlerT.,
SawitzkeJ.,
SergueevK.,
AustinS.2000; A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation. EMBO J 19:6249–6258
CampbellJ. L.,
KlecknerN.1990; E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork. Cell 62:967–979
DemarreG.,
ChattorajD. K.2010; DNA adenine methylation is required to replicate both Vibrio cholerae chromosomes once per cell cycle. PLoS Genet 6:e1000939
DryseliusR.,
IzutsuK.,
HondaT.,
IidaT.2008; Differential replication dynamics for large and small Vibrio chromosomes affect gene dosage, expression and location. BMC Genomics 9:559
DuigouS.,
KnudsenK. G.,
SkovgaardO.,
EganE. S.,
Løbner-OlesenA.,
WaldorM. K.2006; Independent control of replication initiation of the two Vibrio cholerae chromosomes by DnaA and RctB. J Bacteriol 188:6419–6424
DuigouS.,
YamaichiY.,
WaldorM. K.2008; ATP negatively regulates the initiator protein of Vibrio cholerae chromosome II replication. Proc Natl Acad Sci U S A 105:10577–10582
EganE. S.,
FogelM. A.,
WaldorM. K.2005; Divided genomes: negotiating the cell cycle in prokaryotes with multiple chromosomes. Mol Microbiol 56:1129–1138
FiebigA.,
KerenK.,
TheriotJ. A.2006; Fine-scale time-lapse analysis of the biphasic, dynamic behaviour of the two Vibrio cholerae chromosomes. Mol Microbiol 60:1164–1178
FossumS.,
CrookeE.,
SkarstadK.2007; Organization of sister origins and replisomes during multifork DNA replication in Escherichia coli . EMBO J 26:4514–4522
HeidelbergJ. F.,
EisenJ. A.,
NelsonW. C.,
ClaytonR. A.,
GwinnM. L.,
DodsonR. J.,
HaftD. H.,
HickeyE. K.,
PetersonJ. D.other authors2000; DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae . Nature 406:477–483
HiragaS.,
IchinoseC.,
OnogiT.,
NikiH.,
YamazoeM.2000; Bidirectional migration of SeqA-bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coli . Genes Cells 5:327–341
KatayamaT.,
FujimitsuK.,
OgawaT.2001; Multiple pathways regulating DnaA function in Escherichia coli : distinct roles for DnaA titration by the datA locus and the regulatory inactivation of DnaA. Biochimie 83:13–17
KochB.,
MaX. F.,
Løbner-OlesenA.2010; Replication of Vibrio cholerae chromosome I in Escherichia coli : dependence on Dam methylation. J Bacteriol 192:3903–3914
OdsbuI.,
Morigen,
SkarstadK.
2009; A reduction in ribonucleotide reductase activity slows down the chromosome replication fork but does not change its localization. PLoS ONE 4:e7617
OnogiT.,
NikiH.,
YamazoeM.,
HiragaS.1999; The assembly and migration of SeqA–Gfp fusion in living cells of Escherichia coli . Mol Microbiol 31:1775–1782
PearsonG. D. N.,
WoodsA.,
ChiangS. L.,
MekalanosJ. J.1993; CTX genetic element encodes a site-specific recombination system and an intestinal colonization factor. Proc Natl Acad Sci U S A 90:3750–3754
RasmussenT.,
JensenR. B.,
SkovgaardO.2007; The two chromosomes of Vibrio cholerae are initiated at different time points in the cell cycle. EMBO J 26:3124–3131
SlaterS.,
WoldS.,
LuM.,
BoyeE.,
SkarstadK.,
KlecknerN.1995; E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration. Cell 82:927–936
SrivastavaP.,
FeketeR. A.,
ChattorajD. K.2006; Segregation of the replication terminus of the two Vibrio cholerae chromosomes. J Bacteriol 188:1060–1070
SuwantoA.,
KaplanS.1989; Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: presence of two unique circular chromosomes. J Bacteriol 171:5850–5859
TrucksisM.,
MichalskiJ.,
DengY. K.,
KaperJ. B.1998; The Vibrio cholerae genome contains two unique circular chromosomes. Proc Natl Acad Sci U S A 95:14464–14469
WigleyP.,
BurtonN. F.2000; Multiple chromosomes in Burkholderia cepacia and B. gladioli and their distribution in clinical and environmental strains of B. cepacia . J Appl Microbiol 88:914–918
WoodfineK.,
FieglerH.,
BeareD. M.,
CollinsJ. E.,
McCannO. T.,
YoungB. D.,
DebernardiS.,
MottR.,
DunhamI.,
CarterN. P.2004; Replication timing of the human genome. Hum Mol Genet 13:191–202