Construction of Expression Shuttle Vectors for the Haloarchaeon Natrinema sp. J7 Based on Its Chromosomal Origins of Replication

Abstract EN

Haloarchaeon Natrinema sp.

J7, the first reported archaeon harboring both plasmid and chromosome-based temperate viruses, is a useful model for investigating archaeal virus-host and virus-virus interactions.

However, the lack of genetic tools has limited such studies.

On the basis of the automatically replicating sequences of the J7 chromosome and the pyrF marker, we constructed seven vectors, six of which were confirmed to possess replication ability in a pyrF-deletion derivative of J7 (J7-F).

Among these vectors, pFJ1, pFJ4, and pFJ6 could be transformed into the host strain with relatively high efficiency (approximately 103 colony-forming units/μg DNA) and were present at about one copy per chromosome.

These three vectors could be stably maintained in J7-F without selection and were used for heterologous protein expression.

Only pFJ6 was found to be present in the transformed cells in an exclusively episomal, nonintegrated state (one copy per chromosome).

In contrast, some pFJ1 and pFJ4 DNA was probably integrated into the J7-F chromosome.

In addition, pFJ6 was found to be compatible with pYCJ in J7 cells, suggesting that these two vectors could be used for further studies of virus-virus and virus-host interactions.