Search

The obligate intracytoplasmic pathogen Rickettsia prowazekii relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interestingly, the R. prowazekii genome encodes four open reading frames that are highly homologous to the well-characterized ATP/PADP translocase Tlc1. Therefore, by annotation, the R. prowazekii genome encodes a total of five ATP/ADP translocases: Tlc1, Tlc2, Tlc3, Tlc4, and Tlc5. We have confirmed by quantitative reverse transcriptase PCR that mRNAs corresponding to all five tic homologues are expressed in R. prowazekii growing in L-929 cells and have shown their heterologous protein expression in Escherichia coli, suggesting that none of the tic genes are pseudogenes in the process of evolutionary meltdown. However, we demonstrate by heterologous expression in E. coli that only Tlc1 functions as an ATP/ADP transporter. A survey of nucleotides and nucleosides has determined that Tlc4 transports CTP, UTP, and GDP. Intriguingly, although GTP was not transported by Tlc4, it was an inhibitor of CTP and UTP uptake and demonstrated a [K.sub.i] similar to that of GDP. In addition, we demonstrate that Tlc5 transports GTP and GDP. We postulate that Tlc4 and Tlc5 serve the primary function of maintaining intracellular pools of nucleotides for rickettsial nucleic acid biosynthesis and do not provide the cell with nucleoside triphosphates as an energy source, as is the case for Tlc1. Although heterologous expression of Tlc2 and Tlc3 was observed in E. coli, we were unable to identify substrates for these proteins.

The accessibility of the Rickettsia prowazekii ATP/ADP translocase transmembrane domains (TMs) IV-VII and IX-XII to the putative, water-filled ATP translocation pathways is determined. The preexposure of many methanethiosulfonate (MTS)-sensitive mutants in TMs V, VII, X and XI to ATP concentrations 10 times the K(sub m) results in protection from MTS-mediated inhibition, thereby confirming exposure of these TMs to the aqueous ATP translocation pathway.

The obligate intracellular bacterium Rickettsia prowazekii has recently been shown to transport the essential metabolite S-adenosylmethionine (SAM). The existence of such a transporter would suggest that the metK gene, coding for the enzyme that synthesizes SAM, is unnecessary for rickettsial growth. Genome sequencing has revealed that this is the case for the metK genes of the spotted fever group and the Madrid E strain of R. prowazekii, which contain recognizable inactivating mutations. However, several strains of the typhus group rickettsiae possess metK genes lacking obvious mutations. In order to determine if these genes code for a product that retains MAT function, an Escherichia coli metK deletion mutant was constructed in which individual rickettsial metK genes were tested for the ability to complement the methionine adenosyltransferase deficiency. Both the R. prowazekii Breinl and R. typhi Wilmington metK genes complemented at a level comparable to that of an E. coil metK control, demonstrating that the typhus group rickettsiae have the capability of synthesizing as well as transporting SAM. However, the appearance of mutations that affect the function of the metK gene products (a stop codon in the Madrid E strain and a 6-bp deletion in the Breinl strain) provides experimental support for the hypothesis that these typhus group genes, like the more degenerate spotted fever group orthologs, are in the process of gene degradation.

The contribution of transmembrane regions I, II, and III of the Rickettsia prowazekii ATP/ADP translocase to the structure of the putative water-filled ATP translocation channel is determined from the accessibility of hydrophilic, thiol-reactive, methanethiosulfonate reagents to a library of 68 independent cycteine-substitution mutants heterologously expressed in Escherichia coli. Based on the lack of accessibility of charged MTS reagents to the thiol groups in mutants of TM III, it appears that TM II is not exposed to the ATP translocation channel.

The two obligate intracellular alphaproteobacteria Rickettsia prowazekii and Caedibacter caryophilus, a human pathogen and a paramecium endosymbiont, respectively, possess transport systems to facilitate ATP uptake from the host cell cytosol. These transport proteins, which have 65% identity at the amino acid level, were heterologously expressed in Escherichia coli, and their properties were compared. The results presented here demonstrate that the caedibacter transporter had a broader substrate than the more selective rickettsial transporter. ATP analogs with modified sugar moieties, dATP and ddATP, inhibited the transport of ATP by the caedibacter transporter but not by the rickettsial transporter. Both transporters were specific for di- and trinucleotides with an adenine base in that adenosine tetraphosphate, AMP, UTP, CTP, and GTP were not competitive inhibitors. Furthermore, the antiporter nature of both transport systems was shown by the dependence of the efflux of [[[alpha]-.sup.32]P]ATP on the influx of substrate (ATP but not dATP for rickettsiae, ATP or dATP for caedibacter).

Results indicate that the chlamydial glucose-6-phosphate transporter, HPTcp, shows a high degree of sequence similarity to that from Escherichia coli phosphate sensor UhpC than to the UhpT transporter. Data reveal that some amino acid residues are conserved in both UhpC and HPTcp.

Research has been conducted on the membrane proteins. The study of the membrane topology and the permissive sites of the membrane protein expressed in Escherichia coli has been carried out based on the the in vitro transposition of mini-Tn-5 derivatives which bear dual pho-lac reporters and the results are described.

Several experiments have been conducted on non-mitochondrial ATP transport which provide deeper insight into the evolutionary and functional relationships between the plant and bacterial transport proteins. The foremost conclusion that could be drawn from these studies involves the endosymbiotic origins of organelles. It is suggested that mitochondria evolved from a primitive eubacterium that displayed oxidative metabolism and lived within a non-oxidative cell.

The entire genome sequence of the Rickettsia prowazekii intracellular parasite which causes epidemic typhus. The genome consists of 834 protein-coding genes that show similarities to those of mitochondrial genes. The R. prowazekii genome has the highest proportion of non-coding DNA so far seen in a microbial genome, suggesting that it has much closer links to mitochondria than any other microbe investigated.

Genetic transformation of the epidemic typhus-causing intracellular pathogen Ricketssia prowazekii was effected by electroporation. The target gene was rpoB encoding the beta subunit of RNA polymerase. The mutant gene inserted in the transformed Rickettsia was the rifampin-resistant RNA polymerase gene. Transformation was effected through homologous recombination. This is the first report of a genetic system in R. prowazekii.

A research study was conducted to analyze the Rickettsia prowazekii's (R prowazekii) gene organization and genetic transcription. The methods used were recombinant DNA technique and RNA technique. Results revealed that the genes found in R prowazekii are arranged into an operon. However, this gene organization seems different from that of the other bacteria. The open reading frame of the R prowazekii is also dissimilar from that of a gram-positive bacteria's open reading frames.

The sigma factor sigma(super 73) of the obligate intracytoplasmic bacterium Rickettsia prowazekii was overexpressed and purified from Escherichia coli to determine its role in transcriptional stimulation of R. prowazekii RNA polymerase. The R. prowazekii sigma(super 73T) and the 75 kDa fusion protein encoded by the recombinant plasmid were able to bind to core DNA-dependent RNA polymerase of both R. prowazekii and E. coli and to stimulate their interaction with a rickettsial promoter.

The 23S and the 5S rRNA genes in Rickettsia prowazekii are closely connected to each other, whereas the 16S rRNA gene is about 4 kb away from the 23S-5S gene cluster. The sequences flanking the 16S and 23S rRNA genes are different from those observed in a typical eubacteria. The methionyl-tRNA(super Met)(sub f) formyltransferase gene is followed by the 23S-5S unit.

Purification of peptidoglycan from an obligate intracellular bacterium Rickettsia prowazekii reveals D-Ala-D-Ala ligase and alanine racemase processes. Addition of D-alanine prevents D-Cycloserine's inhibition of rickettsial growth in mouse L929 cells.

The copy number of the Rickettsia prowazekii gene encoding 16S rRNA was determined. The first approach used restriction enzyme analysis, which revealed that the genome of R. prowazekii contains only one 16S rRNA gene. The second approach used density comparisons using the seven 16S rRNA fragments of Escherichia coli for comparison. The results also showed that R. prowazekii possesses only one 16S rRNA gene.

Research has been conducted on the etiologic agent of epidemic typhus Rickettsia prowazekii. The expression of Escherichia coli ereB gene in this parasitic bacterium has been performed.

Analysis of the obligate intracytoplasmic Rickettsia prowazeki indicates the presence of transcriptional regulation. The glt promoter in the P2 region of the microbe's deoxyribonucleic acid (DNA) indicated an inverted repeat sequence in the 47 bp region that is located upstream of the glt coding region. This DNA feature is a characteristic of microbes that possess positive transcriptional regulatory mechanisms.

The procaryotic RNA polymerase of the obligate intracellular parasite Rickettsia prowazekii was compared with Escherichia coli's sigma-70, focusing on their behavior during the start of transcription. While E. coli's RNA polymerase-promoter complexes remained stable, R. Prowazekii's complexes were unstable, dissociable and reactive to heparin. Heparin sensitivity was regulated mainly by the R. prowazekii core.

An RNase protection assay aids in the study of the half-life of the RNA transcribed from the Rickettsia prowazekii ATP/ADP translocase gene (tlc) encoding a membrane protein, citrate synthase gene (glt A). The hot-phenol method separates the total RNA, comprising rickettsial RNA and host cell RNA, from the mouse L929 cells infected with rickettsia. In recombinant Escherichia coli strains, the half life of glt A mRNAI is 1.4 plus or minus 0.1 min, and that of tlc mRNA is 2.9 plus or minus 0.1 min.

An efficient method is described for the isolation of highly purified RNA from the obligate intracytoplasmic pathogen Rickettsia prowazekii. This is the first microarray analysis of R. prowazekii by using a model stress response that could also be applied to study other obligate intracellular organisms.

The evaluation of the contribution of transmembrane region VIII of the Rickettsia prowazekii ATP/ADP translocase to the structure of the water filled channel through which ATP is transported is discussed. The results indicate that TM VIII makes a major contribution to the structure of the aqueous translocation pathway, that the accessibility to impermeant thiol reagents is influenced by substrate, and that there is great variation in accessibility to MTS reagents along the length of TM VIII.

Research describes for the first time the existence of a transport mechanism for S-adenosylmethionine in bacteria as exemplified by Rickettsia prowazekii. Results show that the gene encoding S-adenosylmethionine transporter is identified in R. prowazekii.