Abstract
MlrA encoded in the mlr gene cluster, mlrA-mlrB-mlrC-mlrD, appears to be responsible for the initial hydrolytic cleavage of the cyclic microcystin structure, based on the results of gene cluster analysis using E. coli. However, it is unknown whether this protein is solely involved in the initial degrading step in microcystin-degrading bacteria and where the corresponding enzyme is located. Thus, we constructed a gene-specific disruption system to knock out chromosomal mlrA in Sphingopyxis sp. C－1 strain, which we previously reported as a microcystin-degrading bacterium possessing the mlr gene cluster in its chromosome. Disruption of mlrA completely prevented the degradation of cyclic microcystin. This is the first report that MlrA is the sole enzyme responsible for degrading the initial cleavage of cyclic microcystin in the C－1 strain. Moreover, we attached a hexahistidine tag to the C－terminal end of MlrA, i.e., MlrAhis6, to determine the location of MlrA and its functions in the cell. We detected MlrAhis6 band only in the membrane in Western blot with anti-penta-His antibody although we prepared and investigated the membrane and cytosol fractions of the C－1 strain. This suggests that MlrA is a membrane-associated protein and that it degrades microcystin in the periplasm. Thus, we suggest that MlrB and MlrC degrade linear microcystin and its tetrapeptide in the periplasm or cytosol after MlrA degrades cyclic microcystin in the periplasm.
