Mutagenesis of the P2 promoter of the major outer membrane protein gene of Chlamydia trachomatis

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Abstract
On the basis of position from the transcription start site, the P2 promoter of the gene encoding the major outer membrane protein (ompA) of Chlamydia trachomatis consists of a -35 hexamer region of -42 aaaaaga TATACAaa -28 and an unusual, GC-rich -10 hexamer region of -13 tTATCGCt -6. The P2 promoter was analyzed by in vitro transcription of templates containing deletions and site-specific mutations. The 5' extent of P2 was located at bp -42. Replacement of wild-type sequence with two G's at positions -41 and 40, -35 and 34, and -29 and 28 resulted in severely decreased transcription. Additionally, the spacing between the -35 and -10 hexamers could not be shortened without adversely affecting in vitro activity. Substitution of G at position -13, -10, -7, or -6 had little or no effect on transcription, whereas substitution of G at -11 or -12 significantly decreased promoter strength. Triple point mutations which changed the -10 hexamer from TATCGC to TATTAT,TATATT, or TATAAT had little effect on promoter activity. Unlike the partially purified C. trachomatis sigma66-RNA polymerase used in this study, purified Escherichia coli sigma70-RNA polymerase did not recognize the wild-type P2 promoter. Mutant P2 templates with -10 hexamers that resembled the consensus recognition site were transcribed by E. coli holoenzyme in vitro, suggesting that C. trachomatis sigma66-RNA polymerase has special promoter recognition properties not found in E. coli sigma70-holoenzyme.