FIG. 3.

P. aeruginosa PAO1 genes amplified by PCR and inserted into the multiple cloning site of the E. coli-P. aeruginosa shuttle vector pUCPSK or pUCPKS, which differ only by the orientation of their multiple cloning sites (36). The resulting vectors, identified on the right, express the inserted gene(s) from a proximal T7 promoter. The letters K, H, X, E, and S represent the KpnI, HindIII, XbaI, EcoRI, and SacI restriction sites, respectively. Upstream of gatB and gatC, we inserted by PCR the Shine-Dalgarno sequence AGGAGG frequently found in P. aeruginosa, 8 His codons (CAC), and a sequence encoding the factor Xa digestion site Arg-Glu-Gly-Arg (with codons preferentially used in P. aeruginosa). Downstream of gltX and aspS, we inserted a sequence encoding the factor Xa digestion site, 6 His codons, and two stop codons.