Masahiro Tatara

Conjugative gene transfer in marine cyanobacteria: Synechococcus sp., Synechocystis sp. and Pseudanabaena sp.

SummaryVersatility of gene transfer by transconjugation in marine cyanobacteria was demonstrated. In this study, seven different marine cyanobacteria were used as recipient cells. First, transconjugation was carried out using the mobilizable transposon (Tn5) carrying plasmid pSUP1021. Transconjugants were observed in all marine cyanobacteria tested. Second, the broad-host-range vector pKT230 (IncQ) was tested for transconjugation. pKT230 has been successfully transferred in a marine cyanobacterium Synechococcus sp. NKBG15041C, and replicated as an autonomous replicon without alteration in the restriction enzyme pattern. A maximum transfer efficiency of 5.2 × 10−4 transconjugants/recipient cell was observed, when mating was performed on agar plates containing low salinity (0.015 m NaCl) medium. This is the first study to demonstrate gene transfer in marine cyanobacteria via transconjugation.

Foreign gene expression in marine cyanobacteria under pseudo-continuous culture

Foreign gene expression in a marine cyanobacterium, Synechococcus NKBG 15041c, has been carried out in both batch and pseudo-continuous culture, using chloramphenicaol acetyltransferase (CAT) as a model peptide and the broad host range vector pKT230. CAT has been successfully expressed in marine cyanobacteria under kanamycin resistance gene promoter. Pseudo-continuous culture of these recombinant marine cyanobacteria has been done by varying the dilution rate. At a dilution rate between 0.01 to 0.02 h-1, highest productivity was achieved. Under these conditions, long-term pseudo-continuous culture of recombinant marine cyanobacteria was achieved for more than 600 h with CAT productivity 18-fold greater than that observed in batch culture.

Cloning of a marine cyanobacterial promoter for foreign gene expression using a promoter probe vector

A marine cyanobacterial promoter was cloned to allow efficient foreign gene expression. This was carried out using chloramphenicol acetyl transferase (CAT) as a marker protein. For rapid and simple measurement of CAT activity, a method based on a fluorescently labeled substrate was improved by utilizing HPLC equipped with a flowthrough fluorescent spectrophotometer. This method was used in conjunction with a newly constructed promoter probe vector. Cyanobacterial transformants, harboring plasmid containing a cloned 2-kbp marine cyanobacterial genomic fragment, showed a 10-fold higher CAT activity, compared with that achieved using the kanamycin-resistant gene promoter. From the sequence analysis of the cloned fragment, a putative promoter region was found.

Pseudo-continuous culture of marine recombinant cyanobacteria under a light dark cycle

SummaryPseudo-continuous culture of recombinant marine cyanobacteria was carried out under a light dark cycle. As a model foreign gene, the structural gene of chloramphenicol acetyl transferase (CAT) was used. Under a light dark alternate conditions, a steady state cell concentration was observed. However, the level of CAT expression did not show a stationary phase, but oscillated with the light dark cycle. This may be due to the lack of energy for foreign peptide synthesis during dark incubation. This result also indicates the possibility of regulation of foreign gene expression by controlling light illumination.