title: Cloning and expression of the pneumococcal autolysin gene in Escherichia coli.
abstract: A 7.5 kb BclI-fragment of Streptococcus pneumoniae DNA has been cloned in Escherichia coli HB101 using pBR322 as a vector. The new plasmid (pGL30) of 12.0 kb expresses a protein that has been characterized by biochemical, immunological and genetic methods as the inactive form (E-form) of the pneumococcal N-acetyl-muramyl-L-alanyl amidase (EC 3.5.1.28). Our results demonstrate that the E-form is the primary product of the lyt gene of S. pneumoniae. The inactive E-form can be converted to the active C-form in vitro by incubation of the E-form enzyme with choline-containing pneumococcal cell walls at low temperature in a similar way to enzyme production in the homologous system. The production of this protein in E. coli HB101 was 500-fold higher than in the homologous host. E. coli CSR603 containing pGL30 and labeled with [35S]methionine synthesized a 35 kd protein. pGL30 can transform at high frequency an autolysin-defective mutant of S. pneumoniae to the lyt+ phenotype.
authors.forename: E
authors.initials: E
authors.lastname: García
authors.forename: J L
authors.initials: JL
authors.lastname: García
authors.forename: C
authors.initials: C
authors.lastname: Ronda
authors.forename: P
authors.initials: P
authors.lastname: García
authors.forename: R
authors.initials: R
authors.lastname: López
mesh_terms: D000581:Amidohydrolases / Q000235:genetics*; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008970:Molecular Weight; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D013296:Streptococcus pneumoniae / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D004262:DNA Restriction Enzymes; D000581:Amidohydrolases; D009238:N-Acetylmuramoyl-L-alanine Amidase