EXPRESSION CLONING OF DIFFERENT BACTERIAL PHOSPHATASE-ENCODING GENES BY HISTOCHEMICAL SCREENING OF GENOMIC LIBRARIES ONTO AN INDICATOR MEDIUM CONTAINING PHENOLPHTHALEIN DIPHOSPHATE AND METHYL GREEN

A system for expression cloning of bacterial phosphatase-encoding gene s has been developed, and its potential has been investigated. The sys tem is based on histochemical screening of bacterial genomic libraries , constructed in an Escherichia coli multicopy plasmid vector, for pho sphatase-producing clones using an indicator medium (named TPMG) made of Tryptose-Phosphate agar supplemented with the phosphatase substrate phenolphthalein diphosphate and the stain methyl green. To test the p erformance of this system, three genomic libraries were constructed fr om bacterial strains of different species which showed different patte rns of phosphatase activity, and were screened using the TPMG medium. Following a partial screening, three different phosphatase-encoding ge nes (respectively encoding a class A non-specific acid phosphatase, an acid-hexose phosphatase and a non-specific alkaline phosphatase) were shotgun-cloned from the above libraries, indicating that the TPMG-bas ed expression cloning system can be useful for rapid isolation of diff erent bacterial phosphatase-encoding genes.