SUMMARY: An α-amylase gene from has previously been cloned in and shown to direct the synthesis of an enzymically active protein of 60000 Dal (Cornelis , 1982). In one particular host, strain HB101, amylase was found to accumulate in the periplasmic space. To study the processing and the location of the amylase, plasmid pAMY2 was introduced into 188 which is a strain constitutive for alkaline phosphatase, a periplasmic marker, and for β-galactosidase, a cytoplasmic marker. Abnormally large amounts of both α-amylase and β-galactosidase were found in the culture fluid of cells grown in rich medium. Furthermore a severe growth defect was found when cells containing pAMY2 were grown in maltose and glycerol media, while the ability to grow on glucose remained normal. This defect could be reversed by two types of spontaneous mutations. Mutations in the first class are located on the plasmid and correspond to the insertional inactivation of the amylase gene by IS1. Mutations in the second class are located on the host chromosome. These results suggest that the synthesis and export of α-amylase is deleterious to , especially in media containing maltose or glycerol as sole carbon source.


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