Replication of Vibrio cholerae chromosome I in Escherichia coli: dependence on dam methylation

Birgit Koch; Xiaofang Ma; Anders Løbner-Olesen

doi:10.1128/jb.00311-10

Replication of Vibrio cholerae chromosome I in Escherichia coli: dependence on dam methylation

Birgit Koch, Xiaofang Ma, Anders Løbner-Olesen

Functional Genomics

21 Citations (Scopus)

Abstract

We successfully substituted Escherichia coli's origin of replication oriC with the origin region of Vibrio cholerae chromosome I (oriCI(Vc)). Replication from oriCI(Vc) initiated at a similar or slightly reduced cell mass compared to that of normal E. coli oriC. With respect to sequestration-dependent synchrony of initiation and stimulation of initiation by the loss of Hda activity, replication initiation from oriC and oriCI(Vc) were similar. Since Hda is involved in the conversion of DnaA(ATP) (DnaA bound to ATP) to DnaA(ADP) (DnaA bound to ADP), this indicates that DnaA associated with ATP is limiting for V. cholerae chromosome I replication, which similar to what is observed for E. coli. No hda homologue has been identified in V. cholerae yet. In V. cholerae, dam is essential for viability, whereas in E. coli, dam mutants are viable. Replacement of E. coli oriC with oriCI(Vc) allowed us to specifically address the role of the Dam methyltransferase and SeqA in replication initiation from oriCI(Vc). We show that when E. coli's origin of replication is substituted by oriCI(Vc), dam, but not seqA, becomes important for growth, arguing that Dam methylation exerts a critical function at the origin of replication itself. We propose that Dam methylation promotes DnaA-assisted successful duplex opening and replisome assembly at oriCI(Vc) in E. coli. In this model, methylation at oriCI(Vc) would ease DNA melting. This is supported by the fact that the requirement for dam can be alleviated by increasing negative supercoiling of the chromosome through oversupply of the DNA gyrase or loss of SeqA activity.

Original language	English
Journal	Journal of Bacteriology
Volume	192
Issue number	15
Pages (from-to)	3903-14
Number of pages	12
ISSN	0021-9193
DOIs	https://doi.org/10.1128/jb.00311-10
Publication status	Published - Aug 2010

Keywords

Adenosine Triphosphatases/metabolism
Base Sequence
Chromosomes, Bacterial/genetics
DNA Replication/physiology
Escherichia coli/metabolism
Escherichia coli Proteins/genetics
Gene Expression Regulation, Bacterial/physiology
Methylation
Molecular Sequence Data
Replication Origin/genetics
Sequence Alignment
Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics
Vibrio cholerae/genetics

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https://jb.asm.org/content/192/15/3903.full.pdf

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title = "Replication of Vibrio cholerae chromosome I in Escherichia coli: dependence on dam methylation",

abstract = "We successfully substituted Escherichia coli's origin of replication oriC with the origin region of Vibrio cholerae chromosome I (oriCI(Vc)). Replication from oriCI(Vc) initiated at a similar or slightly reduced cell mass compared to that of normal E. coli oriC. With respect to sequestration-dependent synchrony of initiation and stimulation of initiation by the loss of Hda activity, replication initiation from oriC and oriCI(Vc) were similar. Since Hda is involved in the conversion of DnaA(ATP) (DnaA bound to ATP) to DnaA(ADP) (DnaA bound to ADP), this indicates that DnaA associated with ATP is limiting for V. cholerae chromosome I replication, which similar to what is observed for E. coli. No hda homologue has been identified in V. cholerae yet. In V. cholerae, dam is essential for viability, whereas in E. coli, dam mutants are viable. Replacement of E. coli oriC with oriCI(Vc) allowed us to specifically address the role of the Dam methyltransferase and SeqA in replication initiation from oriCI(Vc). We show that when E. coli's origin of replication is substituted by oriCI(Vc), dam, but not seqA, becomes important for growth, arguing that Dam methylation exerts a critical function at the origin of replication itself. We propose that Dam methylation promotes DnaA-assisted successful duplex opening and replisome assembly at oriCI(Vc) in E. coli. In this model, methylation at oriCI(Vc) would ease DNA melting. This is supported by the fact that the requirement for dam can be alleviated by increasing negative supercoiling of the chromosome through oversupply of the DNA gyrase or loss of SeqA activity.",

keywords = "Adenosine Triphosphatases/metabolism, Base Sequence, Chromosomes, Bacterial/genetics, DNA Replication/physiology, Escherichia coli/metabolism, Escherichia coli Proteins/genetics, Gene Expression Regulation, Bacterial/physiology, Methylation, Molecular Sequence Data, Replication Origin/genetics, Sequence Alignment, Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics, Vibrio cholerae/genetics",

author = "Birgit Koch and Xiaofang Ma and Anders L{\o}bner-Olesen",

year = "2010",

month = aug,

doi = "10.1128/jb.00311-10",

language = "English",

volume = "192",

pages = "3903--14",

journal = "Journal of Bacteriology",

issn = "0021-9193",

publisher = "American Society for Microbiology",

number = "15",

}

TY - JOUR

T1 - Replication of Vibrio cholerae chromosome I in Escherichia coli

T2 - dependence on dam methylation

AU - Koch, Birgit

AU - Ma, Xiaofang

AU - Løbner-Olesen, Anders

PY - 2010/8

Y1 - 2010/8

N2 - We successfully substituted Escherichia coli's origin of replication oriC with the origin region of Vibrio cholerae chromosome I (oriCI(Vc)). Replication from oriCI(Vc) initiated at a similar or slightly reduced cell mass compared to that of normal E. coli oriC. With respect to sequestration-dependent synchrony of initiation and stimulation of initiation by the loss of Hda activity, replication initiation from oriC and oriCI(Vc) were similar. Since Hda is involved in the conversion of DnaA(ATP) (DnaA bound to ATP) to DnaA(ADP) (DnaA bound to ADP), this indicates that DnaA associated with ATP is limiting for V. cholerae chromosome I replication, which similar to what is observed for E. coli. No hda homologue has been identified in V. cholerae yet. In V. cholerae, dam is essential for viability, whereas in E. coli, dam mutants are viable. Replacement of E. coli oriC with oriCI(Vc) allowed us to specifically address the role of the Dam methyltransferase and SeqA in replication initiation from oriCI(Vc). We show that when E. coli's origin of replication is substituted by oriCI(Vc), dam, but not seqA, becomes important for growth, arguing that Dam methylation exerts a critical function at the origin of replication itself. We propose that Dam methylation promotes DnaA-assisted successful duplex opening and replisome assembly at oriCI(Vc) in E. coli. In this model, methylation at oriCI(Vc) would ease DNA melting. This is supported by the fact that the requirement for dam can be alleviated by increasing negative supercoiling of the chromosome through oversupply of the DNA gyrase or loss of SeqA activity.

AB - We successfully substituted Escherichia coli's origin of replication oriC with the origin region of Vibrio cholerae chromosome I (oriCI(Vc)). Replication from oriCI(Vc) initiated at a similar or slightly reduced cell mass compared to that of normal E. coli oriC. With respect to sequestration-dependent synchrony of initiation and stimulation of initiation by the loss of Hda activity, replication initiation from oriC and oriCI(Vc) were similar. Since Hda is involved in the conversion of DnaA(ATP) (DnaA bound to ATP) to DnaA(ADP) (DnaA bound to ADP), this indicates that DnaA associated with ATP is limiting for V. cholerae chromosome I replication, which similar to what is observed for E. coli. No hda homologue has been identified in V. cholerae yet. In V. cholerae, dam is essential for viability, whereas in E. coli, dam mutants are viable. Replacement of E. coli oriC with oriCI(Vc) allowed us to specifically address the role of the Dam methyltransferase and SeqA in replication initiation from oriCI(Vc). We show that when E. coli's origin of replication is substituted by oriCI(Vc), dam, but not seqA, becomes important for growth, arguing that Dam methylation exerts a critical function at the origin of replication itself. We propose that Dam methylation promotes DnaA-assisted successful duplex opening and replisome assembly at oriCI(Vc) in E. coli. In this model, methylation at oriCI(Vc) would ease DNA melting. This is supported by the fact that the requirement for dam can be alleviated by increasing negative supercoiling of the chromosome through oversupply of the DNA gyrase or loss of SeqA activity.

KW - Adenosine Triphosphatases/metabolism

KW - Base Sequence

KW - Chromosomes, Bacterial/genetics

KW - DNA Replication/physiology

KW - Escherichia coli/metabolism

KW - Escherichia coli Proteins/genetics

KW - Gene Expression Regulation, Bacterial/physiology

KW - Methylation

KW - Molecular Sequence Data

KW - Replication Origin/genetics

KW - Sequence Alignment

KW - Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics

KW - Vibrio cholerae/genetics

U2 - 10.1128/jb.00311-10

DO - 10.1128/jb.00311-10

M3 - Journal article

C2 - 20511501

SN - 0021-9193

VL - 192

SP - 3903

EP - 3914

JO - Journal of Bacteriology

JF - Journal of Bacteriology

IS - 15

ER -

Replication of Vibrio cholerae chromosome I in Escherichia coli: dependence on dam methylation

Abstract

Keywords

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