rctB mutations that increase copy number of Vibrio cholerae oriCII in Escherichia coli
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rctB mutations that increase copy number of Vibrio cholerae oriCII in Escherichia coli. / Koch, Birgit; Ma, Xiaofang; Løbner-Olesen, Anders.
I: Plasmid, Bind 68, Nr. 3, 11.2012, s. 159-169.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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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
VL - 68
SP - 159
EP - 169
JO - Plasmid
JF - Plasmid
SN - 0147-619X
IS - 3
ER -
ID: 200971327