FIG. 1.

Constructs for GFP expression. (A) pGB5P1. The gfp-mutant 2 gene (2) was cloned as a BamHI-EcoRI restriction fragment into the pBBR1MCS-2 vector (15). A Sau3A restriction fragment that encodes a constitutive B. pertussis promoter was cloned upstream to control gfp expression. (B) pCW245-1. Nucleotides 1 to 251 from the B. pertussis cpm 10 (5) promoter were amplified by PCR, and the product was digested with PstI and HaeIII and then cloned into pGFPuv to control gfp expression, generating pCW211-6. A PstI restriction fragment (nucleotides 11810 to 13025) from the end of the ptl operon was cloned into pUW2139 [pBluescript SK(+) containing gent/oriT], and the resulting construct, pCW204-1, was digested with ApaI and ligated with pCW211-6 to generate pCW245-1. Plasmid CW245-1 was introduced into bacteria by triparental mating as previously described (19), and transformants were selected on BGA, nalidixic acid, and gentamicin (30 μg/ml). Abbreviations: mob, mobilizable gene; rep, plasmid replication; gfp mut2, green fluorescent protein mutant 2; P1, B. pertussis constitutive promoter; kan, kanamycin; cpm 10, chaperonin 10 (B. pertussis GroES homologue); ptl, pertussis toxin liberation; gent/oriT, gentamicin/origin of transfer; amp, ampicillin.