The Rep20 replication initiator from the pAG20 plasmid of Acetobacter aceti

Martin Babič, Zuzana Rešková, Juraj Bugala, Viera Cimová, Peter Grones*, Jozef Grones

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

5 Citations (Scopus)


In the previously isolated pAG20 plasmid from the Acetobacter aceti CCM3610 strain, the Rep20 protein was characterized as a main replication initiator. The pAG20 plasmid origin was localized in the vicinity of the rep20 gene and contained two 21-nucleotide-long iteron sequences, two 13-nucleotide-long direct repeats, and a DnaA-binding site. Electrophoretic mobility shift assay and nonradioactive fragment analysis confirmed that the Rep20 protein interacted with two direct repeats (5′- TCCAAATTTGGAT′-3′) and their requirement during plasmid replication was verified by mutagenesis. Although the association could not be validated of the DnaA protein of from the host cells of Escherichia coli with the plasmid-encoded replication initiator that usually occurs during replication initiation, Rep20 was able to form dimeric structures by which it could bind the sequence of the rep20 gene and autoregulate its own expression. Targeted mutagenesis of the Rep20 protein revealed the importance of the third α-helix and 63Lys, specifically during DNA binding. The second, closely adjacent β-sheet also took part in this process in which 52Asn played a significant role.
Original languageEnglish
Pages (from-to)1-11
Number of pages11
JournalMolecular Biotechnology
Issue number1
Publication statusPublished - Jan 2014
Externally publishedYes


  • Acetobacter aceti
  • Bacterial plasmid
  • DNA fragment analysis
  • Electrophoretic mobility shift assay
  • Protein purification
  • Replication protein


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