Cloning and expression of the pneumococcal autolysin gene in Escherichia coli |
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Authors: | Ernesto García José-Luís García Concepción Ronda Pedro Garcá and Rubens López |
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Institution: | (1) Instituto de Immunología y Biología Microbiana, Velázquez 144, SP-28006 Madrid;(2) Antibióticos, S.A. Bravo Murillo 38, SP-28015 Madrid, Spain |
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Abstract: | Summary A 7.5 kb BclI-fragment of Streptococcs 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. |
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