Identification of the gene for disaggregatase fromMethanosarcina mazei
The gene sequences encoding disaggregatase (Dag), the enzyme responsible for dispersion of cell aggregates ofMethanosarcina mazeito single cells, were determined for three strains ofM. mazei(S-6T, LYC and TMA). Thedaggenes of the three strains were 3234 bp in length and had almost the same sequences with 97% amino acid sequence identities. Dag was predicted to comprise 1077 amino acid residues and to have a molecular mass of 120 kDa containing three repeats of the DNRLRE domain in the C terminus, which is specific to the genusMethanosarcinaand may be responsible for structural organization and cell wall function. Recombinant Dag was overexpressed inEscherichia coliand preparations of the expressed protein exhibited enzymatic activity. The RT-PCR analysis showed thatdagwas transcribed to mRNA inM. mazeiLYC and indicated that the gene was expressed in vivo. This is the first time the gene involved in the morphological change ofMethanosarcinaspp. from aggregate to single cells has been identified.