Sequence analysis and minimal replicon determination of a new haloarchaeal plasmid pHF2 isolated from Haloferax sp. strain Q22

• The smallest plasmid (2520 bp) in the genus Haloferax
• The Rep protein, along with the DSO, is sufficient to support plasmid replication.
• The smallest replicon (~ 1 kb) was determined for shuttle vector construction.
• A new plasmid derived from subterranean halite deposit

A new cryptic plasmid, pHF2 (2520 bp), was isolated from Haloferax sp. strain Q22 ( CGMCC 1.15317), a haloarchaeal strain living in a subterranean halite deposit. Sequence analysis revealed that it is the smallest plasmid in the genus Haloferax so far, and three syntropic open reading frames (ORF1, ORF2, and ORF3) were identified on the same strand. ORF1 encodes a putative replication initiation protein (Rep). Three typical motifs (I, II, and III) were presented in the Rep proteins of rolling-circle replicating (RCR) plasmids. The amino acid sequence of the Rep protein is very similar to that of another haloarchaeal plasmid pNB101 in Natronobacterium sp. AS-7091 (coverage 97%, identity 56%). The minimal replicon (~ 1000 bp) of pHF2 was determined through the construction of a series of truncated plasmids. Interestingly, we also found that the incomplete rep gene still can drive plasmid replication. This plasmid has provided another valuable extra-chromosomal genetic resource, and deepened our knowledge in DNA replication.