The presence of different tryptophan codon contents within paralogous genes in C. pneumoniae affects their transcription during IFN-γ-mediated tryptophan limitation. Nucleic acid samples were collected from untreated (solid symbols) and IFN-γ-treated (open symbols) C. pneumoniae-infected HEp2 cells at 24 and 48 h postinfection. Transcript levels for the indicated genes were measured by RT-qPCR and normalized to genomic DNA (nanograms of cDNA/gDNA). (A) Quantification of transcript levels for groEL paralogues, where groEL_1 encodes 0 Trp codons, groEL_2 encodes 2 Trp codons, and groEL_3 encodes 1 Trp codon. (B) Western blot measurement of chlamydial Hsp60_1 (i.e., GroEL_1) from untreated and IFN-γ-treated C. pneumoniae-infected cultures. Protein loading was normalized for C. pneumoniae genomic DNA levels. Densitometry readings are displayed below the bands. (C) Quantification of transcript levels for clpP paralogues where clpP_1 encodes 3 Trp codons and clpP_2 encodes 0 Trp codons. (D) Quantification of transcript levels for rsbV paralogues where neither encodes Trp codons. All transcript data are the average from at least 2 experiments with standard deviations displayed. *, P ≤ 0.05 for 48-hpi IFN-γ versus 24-hpi IFN-γ.