title: Cloning and expression of the phospho-beta-galactosidase gene of Staphylococcus aureus in Escherichia coli.
abstract: The phospho-beta-galactosidase gene of Staphylococcus aureus was cloned in Escherichia coli. This was done by first isolating a staphylococcal transposon Tn551-induced mutant which rendered phospho-beta-galactosidase synthesis partially constitutive because of an insertion nearby this lac structural gene. This allowed selection in E. coli of chimeric plasmids which expressed the erythromycin resistance determinant of Tn551. A 26-kilobase (kb) BamHI insert in plasmid pBR322 was isolated which encoded phospho-beta-galactosidase, as determined by phospho-beta-galactosidase activity measurements. Maxicell experiments showed the presence of 56-, 13.5-, and 31-kilodalton proteins encoded by the staphylococcal DNA. The presence of the 56-kilodalton protein correlated with phospho-beta-galactosidase activity and corresponded in molecular weight to the reported value for the purified enzyme. The nature of the other proteins is unknown. Phospho-beta-galactosidase was apparently expressed in E. coli by a promoter contained within a 2.1-kb EcoRI chromosomal DNA fragment. This fragment, when inserted into a chloramphenicol acetyl transferase promoter detection plasmid, was transcriptionally active in both E. coli and Bacillus subtilis but was much more active in the latter host.
mesh_terms: D000123:Acetyltransferases / Q000235:genetics; D002678:Chimera; D015500:Chloramphenicol O-Acetyltransferase; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000032:analysis; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI; D004352:Drug Resistance, Microbial; D004917:Erythromycin / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D005696:Galactosidases / Q000235:genetics*; D005786:Gene Expression Regulation; D005796:Genes; D006026:Glycoside Hydrolases; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004917:Erythromycin; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI; D005696:Galactosidases; D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; C022361:6-phospho-beta-galactosidase