FIG. 1.

Organization of the clpX operon in S. pneumoniae R6 and construction of a nonpolar replacement of the clpX reading frame with ermAM (Erm^r) (A) in a strain containing an ectopic copy of clpX⁺ under the control of the fucose-inducible P_fcsK promoter located in the bgaA locus (B) (see text and Table 1 for details). Genes and predicted directions of transcription are depicted by arrows (drawn to scale). The lytC and trxB genes likely mark the boundaries of the clpX region based on the large intergenic region and the direction of transcription, respectively. The shaded regions depict the flanking genes surrounding clpX, and the hatched region corresponds to the ermAM open reading frame. P_c-tetL imparts constitutive resistance to 0.25 μg of tetracycline per ml and was used as a selection marker. T1T2 indicates transcription terminators to protect regulated expression from P_fcsK.