rctB mutations that increase copy number of Vibrio cholerae oriCII in Escherichia coli

TY - JOUR

T1 - rctB mutations that increase copy number of Vibrio cholerae oriCII in Escherichia coli

AU - Koch, Birgit

AU - Ma, Xiaofang

AU - Løbner-Olesen, Anders

N1 - Copyright © 2012 Elsevier Inc. All rights reserved.

PY - 2012/11

Y1 - 2012/11

N2 - RctB serves as the initiator protein for replication from oriCII, the origin of replication of Vibrio cholerae chromosome II. RctB is conserved between members of Vibrionaceae but shows no homology to known replication initiator proteins and has no recognizable sequence motifs. We used an oriCII based minichromosome to isolate copy-up mutants in Escherichia coli. Three point mutations rctB(R269H), rctB(L439H) and rctB(Y381N) and one IS10 insertion in the 3'-end of the rctB gene were obtained. We determined the maximal C-terminal deletion that still gave rise to a functional RctB protein to be 165 amino acids. All rctB mutations led to decreased RctB-RctB interaction indicating that the monomer is the active form of the initiator protein. All mutations also showed various defects in rctB autoregulation. Loss of the C-terminal part of RctB led to overinitiation by reducing binding of RctB to both rctA and inc regions that normally serve to limit initiation from oriCII. Overproduction of RctB(R269H) and RctB(L439H) led to a rapid increase in oriCII copy number. This suggests that the initiator function of the two mutant proteins is increased relative to the wild-type.

AB - RctB serves as the initiator protein for replication from oriCII, the origin of replication of Vibrio cholerae chromosome II. RctB is conserved between members of Vibrionaceae but shows no homology to known replication initiator proteins and has no recognizable sequence motifs. We used an oriCII based minichromosome to isolate copy-up mutants in Escherichia coli. Three point mutations rctB(R269H), rctB(L439H) and rctB(Y381N) and one IS10 insertion in the 3'-end of the rctB gene were obtained. We determined the maximal C-terminal deletion that still gave rise to a functional RctB protein to be 165 amino acids. All rctB mutations led to decreased RctB-RctB interaction indicating that the monomer is the active form of the initiator protein. All mutations also showed various defects in rctB autoregulation. Loss of the C-terminal part of RctB led to overinitiation by reducing binding of RctB to both rctA and inc regions that normally serve to limit initiation from oriCII. Overproduction of RctB(R269H) and RctB(L439H) led to a rapid increase in oriCII copy number. This suggests that the initiator function of the two mutant proteins is increased relative to the wild-type.

KW - Chromosomes, Bacterial/genetics

KW - DNA Helicases/genetics

KW - DNA Replication/genetics

KW - Escherichia coli/genetics

KW - Gene Expression Regulation, Bacterial

KW - Mutation

KW - Origin Recognition Complex/genetics

KW - Point Mutation

KW - Replication Origin/genetics

KW - Trans-Activators/genetics

KW - Vibrio cholerae/genetics

U2 - 10.1016/j.plasmid.2012.03.003

DO - 10.1016/j.plasmid.2012.03.003

M3 - Journal article

C2 - 22487081

SN - 0147-619X

VL - 68

SP - 159

EP - 169

JO - Plasmid

JF - Plasmid

IS - 3

ER -