Identification of cis- and trans-acting elements in pHW126, a representative of a novel group of rolling circle plasmids.
pHW126, pIGRK, pIGMS31 and pRAO1 are the only known members of a novel and as yet uncharacterised family of rolling circle plasmids. pHW126 contains only two open reading frames, of which one shows homology to pMV158-family mobilisation proteins. Here we provide evidence that the second open reading frame encodes a replication protein (Rep). Mutation or deletion of this gene resulted in replication deficient constructs, but providing functional Rep from a compatible vector rescued these constructs, indicating that Rep acts in trans. An approximately 300 bp cis-acting region representing the origin of replication was identified upstream of the rep gene. The origin was identified to be composed of three parts: an accessory region, a conserved stretch and four perfect tandem repeats. The two latter elements were essential for replication. Constructs with a deletion of the accessory region could still replicate, but their loss rate was high, indicating that the accessory region is necessary for plasmid maintenance under non-selective conditions. Interestingly, pHW126 could replicate in all Enterobacteriaceae tested while Agrobacterium tumefaciens and Pseudomonas syringae were inappropriate hosts. Thus, pHW126 seems to have a rather limited host range.
Rozhon
Wilfried
W
Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, 1030 Vienna, Austria. wilfried.rozhon@univie.ac.at
Khan
Mamoona
M
Petutschnig
Elena
E
Poppenberger
Brigitte
B
D001483:Base Sequence; D004261:DNA Replication / Q000235:genetics; D004270:DNA, Circular / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics; D058507:Host Specificity / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D020638:Rahnella / Q000201:enzymology / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D018741:Replication Origin / Q000235:genetics
D004270:DNA, Circular; D003850:Deoxyribonuclease I