Identification of a potential membrane-targeting region of the replication initiator protein (TrfA) of broad-host-range plasmid RK2.
Plasmid RK2 codes for two species of the replication initiator protein TrfA (33 and 44 kDa). Both polypeptides are strongly associated with membrane fractions of Escherichia coli host cells (W. Firshein and P. Kim, Mol. Microbiol. 23, 1-10, 1997). We investigated the role of a 12-amino-acid hydrophobic region (HR) in the membrane association of TrfA. Epitope-tagged polypeptide fragments of TrfA that contained HR were expressed and found to be associated with membrane fractions. Site-directed mutagenesis of trfA revealed that changes of specific amino acids in HR can affect both TrfA association with the membrane and its ability to support replication of an RK2 oriV plasmid in vivo. These results are consistent with the hypothesis that membrane association of TrfA is functionally relevant and that the HR region of TrfA is involved in membrane association and DNA replication in vivo.
Kim
P D
PD
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459, USA.
Rosche
T M
TM
Firshein
W
W
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; C067405:TrfA protein, E coli