Construction and genetic analyses of mutant for rrnB operon in extremely halophilic archaeon, Haloarcula japonica

概要

論文の詳細を見る
Construction of plasmid for rrnB operon disruption of extremely halophilic archaeon, Haloarcula japonica, was carried out. The shuttle vector, pWL102 which is replicated in both Escherichia coli and H. japonica, was used to construct the gene-disruptant plasmid. The insert for gene disruption with homologous recombination was prepared to amplify fragments of upstream and downstream region of rrnB operon using SOE-PCR approach. Prepared plasmid, pDRB was transformed to introduce into spheroplasted cell of H. japonica using polyethylene glycol. Twice screenings were performed to obtain rrnB disruptant. First, the screening with mevinolin (Mev) was done to generate mevinolin-resistant transformant through the first homologous recombination, and then, the medium without mevinolin was used to screen to obtain complete rrnB disruptant with second homologous recombination. The isolated microorganism was designated as H. japonica strain TRU002. In addition, growth of wild and mutant strain, was measured to calculate doubling time of cell division. The measurement of cell growth showed rrnB disruption (doubling time; 22.1hr) affects microbial growth, compared with wild type strain (doubling time; 16.7hr). The colony formation was also affected in the generation time. The reverse-genetics approach using gene-disruption plasmid is definitely useful to understand the effects of cell growth of H. japonica.