The obligate intracellular parasite, Rickettsia prowazekii, is a slow-growing bacterium with a doubling time of about 10 h. In the present study, DNA and RNA were obtained from the rickettsiae by two independent methods, i.e. simultaneous isolation of DNA and RNA from the same sample by phenol:chloroform extraction and CsCI gradient centrifugation. In addition, ribosomal RNA was obtained by sedimentation of partially purified ribosomes from the rickettsiae. The results demonstrated that, after correction for the cell volumes, the concentrations of stable RNA and ribosomes in R. prowazekii, a slow-growing organism, were about 62 fg micron-3 and 17,000 per micron3, respectively, which were very similar (66 fg micron-3 and 21,000 per micron3) to those in Escherichia coli with a generation time of 40 min. However, on a per cell basis, R. prowazekii had 5.6 fg of RNA and 1500 ribosomes per cell, which was only about 8% of the amount of both stable RNA (71.2 fg) and ribosomes (24,000) per cell as was found in E. coli. These results indicated that R. prowazekii possesses a ribosome concentration greater than might have been predicted from its slow growth rate. This high concentration of ribosomes could be due to a large population of nonfunctioning ribosomes, a low efficiency of amino acid production, or a high rate of protein turnover. However, this study also demonstrated that the rickettsiae have very limited protein turnover. Knowledge of the kinetics and control mechanisms for protein synthesis in R. prowazekii remains to be established to determine the logic of the extra rickettsial ribosomes.

In the present study, peptidoglycan from Rickettsia prowazekii, an obligate intracellular bacterium, was purified. The rickettsial peptidoglycan is like that of gram-negative bacteria; that is, it is sodium dodecyl sulfate insoluble, lysozyme sensitive, and composed of glutamic acid, alanine, and diaminopimelic acid in a molar ratio of 1.0:2.3:1.0. The small amount of lysine found in the peptidoglycan preparation suggests that a peptidoglycanlinked lipoprotein(s) may be present in the rickettsiae. D-Cycloserine, a D-alanine analog which inhibits the biosynthesis of bacterial cell walls, prevented rickettsial growth in mouse L929 cells at a high concentration and altered the morphology of the rickettsiae at a low concentration. These effects were prevented by the addition of D-alanine. This suggests that R. prowazekii contains D-alanine in the peptidoglycan and has D-Ala-D-Ala ligase and alanine racemase activities.Rickettsia prowazekii, the etiological agent of epidemic typhus, is an obligate intracellular bacterium with typical gramnegative bacterial morphology (31, 32). In thin sections of R. prowazekii, a cytoplasmic membrane and an outer membrane defining a prominent periplasmic space are routinely observed (2,3,15). In most bacteria, a defined structural element in the cell envelope, peptidoglycan, maintains the integrity and the shape of the bacterium (27). It is not established whether R. prowazekii, or any other member of the genus, has peptidoglycan. As small obligate intracellular parasites, growing in eucaryotic cytoplasm and migrating from host cell to host cell in extracellular biological fluids, rickettsiae may not be challenged by the osmotic stresses faced by free-living bacteria. It has been reported that peptidoglycan appears to be either absent or highly deficient in Rickettsia tsutsugamushi (1). Indirect evidence indicates that R. prowazekii contains peptidoglycan: muramic acid and diaminopimelic acid (DAP) have been found in cell lysates of rickettsiae (10, 18), and penicillin inhibits growth and induces the formation of spheroplasts in cultures of R. prowazekii (33). However, some bacteria contain DAP as a precursor of lysine but have lysine, not DAP, in the peptidoglycan (4, 24). Moreover, the amino acid composition in the rickettsial cell wall preparations used in previous studies (9, 10, 18) was not much different from that in the whole-cell extract. It is unlikely that 15 or more amino acids represent the composition of the rickettsial peptidoglycan. In the present study, we demonstrated that R. prowazekii has sodium dodecyl sulfate (SDS)-insoluble, lysozyme-sensitive peptidoglycan and that the purified peptidoglycan has an amino acid profile, including DAP, similar to that of Escherichia coli peptidoglycan. To the best of our knowledge, this is the first isolation and characterization of the amino acid composition of the peptidoglycan of either an obligate intracellular parasite or a member of the alpha-purple group of eubacteria.Isolation of peptidoglycan from R. prowazekii. The metho...

The obligate intracellular parasite, Rickettsia prowazekii, is a slowly growing bacterium with a doubling time of 8 to 12 h. The copy number of the 16S rRNA gene in the rickettsial chromosome was determined to be one. Genomic DNA from R. prowazekii was digested either by a variety of restriction enzymes known not to cut at any site in the rickettsial 16S rRNA gene or by a combination of these noncutting enzymes and SmaI, which cuts the gene only once. Only one DNA fragment in these digests hybridized to a biotinylated probe containing a portion of the rickettsial 16S rRNA gene. Moreover, the density of the rickettsial 16S rRNA gene fragment after hybridization was equal to the density of each of the seven 16S rRNA gene fragments in Escherichia coli.

By transducing an apoptotic signal in immune effector cells, Fas has been directly implicated in the control of immunological activity. Expression and functional results, however, have also suggested a role for Fas in regulating cell turnover in specific epithelial populations. To characterize factors responsible for Fas expression in epithelial cells, approximately 3 kb of the 5' flanking region of the mouse Fas gene was isolated. By rapid amplification of cDNA ends and primer extension, transcriptional start sites were identified within 50 bp upstream of the translation start site. Transient transfection of promoter-luciferase constructs in a mouse lung epithelial cell line, MLE-15, localized promoter activity to the first 77 bp of upstream sequence. By using a 60 bp DNA probe (-18 to -77) in electrophoretic mobility-shift assays, three shifted complexes were found. Incubation with excess cold Sp1 oligonucleotide or an anti-Sp3 antibody inhibited complex formation. Site-directed mutagenesis of the Sp1 site resulted in 60-70% loss of promoter activity. In Drosophila SL-2 cells, promoter activity was markedly increased by co-transfection of an Sp3 expression construct. These results show that the Sp3 protein is involved in regulating Fas gene expression in lung epithelial cells.

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...