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 genes has been developed, and its potential has been investigated. The system is based on histochemical screening of bacterial genomic libraries, constructed in an Escherichia coli multicopy plasmid vector, for phosphatase-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 performance of this system, three genomic libraries were constructed from bacterial strains of different species which showed different patterns of phosphatase activity, and were screened using the TPMG medium. Following a partial screening, three different phosphatase-encoding genes (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-based expression cloning system can be useful for rapid isolation of different bacterial phosphatase-encoding genes.
Riccio
M L
ML
Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, Siena, Italy.
Rossolini
G M
GM
Lombardi
G
G
Chiesurin
A
A
Satta
G
G
D000135:Acid Phosphatase / Q000235:genetics*; D001419:Bacteria / Q000187:drug effects / Q000201:enzymology* / Q000302:isolation & purification; D001431:Bacteriological Techniques; D003001:Cloning, Molecular / Q000379:methods*; D004396:Coloring Agents / Q000378:metabolism; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008739:Methyl Green / Q000378:metabolism; D010635:Phenolphthaleins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism*; D013194:Staining and Labeling
D004396:Coloring Agents; D003470:Culture Media; D010635:Phenolphthaleins; D011993:Recombinant Fusion Proteins; D008739:Methyl Green; D000135:Acid Phosphatase