Transcriptional analysis of the 16s rRNA gene in Rickettsia prowazekii

Abstract

The control of rRNA synthesis in the etiological agent of epidemic typhus, Rickettsia prowazekii, a slowly growing obligate intracytoplasmic bacterium, was investigated. Transcription of the rickettsial 16S rRNA gene (rrs), of which there is only a single copy, was controlled by a single promoter region, and the site for the initiation of transcription (base A) was found 117 bp upstream of the rrs coding region for the mature product. The promoter region contained an Escherichia coli promoter-like sequence, TTGACA-N 17 -TATAAC, centered 139 bp upstream of the coding region for the mature product. To investigate whether transcription of the rickettsial rrs responds to amino acid starvation conditions, total RNA was isolated from R. prowazekii-infected mouse L929 cells with or without methionine starvation. The level of newly synthesized 16S rRNA precursors in R. prowazekii, as analyzed by ribonuclease protection assays, decreased significantly after methionine starvation for 6 h and then recovered within 12 h after the addition of methionine. The chemical half-lives of the 16S rRNA precursors in the methionine-starved rickettsiae did not differ significantly from those in the normal rickettsiae. These results suggest that R. prowazekii regulates transcription of the rrs in response to amino acid starvation conditions. Rickettsia prowazekii, the etiological agent of epidemic typhus, is an obligate intracytoplasmic bacterium with a typical gram-negative bacterial morphology (24,25). The growth rate of the rickettsiae (about 10 h per generation) (26, 27) is considered low in comparison with the growth rates of facultative bacteria. In general, the rate of rRNA synthesis determines the rate of ribosome synthesis, is responsive to the nutritional environment, and is under growth rate-dependent control (9,10,14). In Escherichia coli, seven redundant rRNA (rrn) gene operons, two tandem promoters for each of the operons, and A/T-rich upstream sequences are considered largely responsible for the high rate of rRNA transcription (10). The R. prowazekii genome has only one 16S rRNA gene (rrs) and only one 23S rRNA gene, and they are at distinct loci (1,16). No information on the upstream sequence and the control of 16S rRNA synthesis in members of the genus Rickettsia was available prior to this study, although the sequence of the mature 16S rRNA had been determined by PCR analysis (23).In the present study, we cloned, sequenced and functionally characterized the upstream promoter region of the rickettsial rrs. We also investigated whether R. prowazekii controls 16S rRNA production in response to amino acid starvation. An understanding of the control of rRNA production could provide valuable insight into global regulatory mechanisms in these obligate intracytoplasmic parasites. MATERIALS AND METHODSConstruction of plasmids. The upstream region and 5Ј end of the coding portion of the rickettsial rrs was cloned into pBluescript SK (Stratagene, La Jolla, Calif.) from the genomic DNA of R. prowazekii. The rickettsial genomic DNA...

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Regulation of whole bacterial pathogen transcription within infected hosts

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