Construction of a broad-host-range pneumococcal promoter-probe plasmid

A promoter-probe plasmid, pLSE4, containing the promoterless lytA gene that encodes the major pneumococcal autolysin, was developed to isolate and characterize nucleotide sequences of Streptococcus pneumoniae involved in transcriptional regulation. This vector was derived from the broad-host-range plasmid pLS1 and is suitable for the transformation of Gram- and Gram+ bacteria. An array of unique restriction sites was placed upstream from the lytA coding region. Pneumococcal promoters can be screened from random DNA fragments cloned in these sites for the ability to direct the expression of the autolysin in transformed autolysin-deficient pneumococcal cells. Transformants showing a Lyt+ phenotype were selected on agar plates using a simple filter technique. Relative promoter strength was determined by direct assay of the cell wall lytic activity in cell extracts.