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Methionine aminopeptidase (MetAp) enzymes catalyze the post-translational removal of the initiator methionine residue in newly synthesized proteins, a process that is often essential in the maturation of proteins. Consequently, these enzymes serve as important targets for drug development. Rickettsia prowazekii (Rp) is an obligate coccobacillus and the causative agent of the louse-borne epidemic typhus and despite adequate treatment causes a latent infection. This research aimed to identify potential anti-rickettsial agents by screening 400 compounds from the MMV Pandemic Response Box against RpMetAp1. Overall, 19 compounds were identified that possessed IC 50 values from 10 µM to 340 nM. The most potent inhibitor was MMV 1580488 (17), which was observed to have an IC 50 of 340 nM. The selected hits serve as chemical leads that can be used for the development of potent inhibitors of the RpMetAp1 enzyme., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Timothy Hagen reports equipment, drugs, or supplies was provided by Medicines for Malaria Venture. Timothy Hagen reports equipment, drugs, or supplies was provided by Seattle Structural Genomics Center for Infectious Disease. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Methionine aminopeptidase (MetAP) is a dinuclear metalloprotease responsible for the cleavage of methionine initiator residues from nascent proteins. MetAP activity is necessary for bacterial proliferation and is therefore a projected novel antibacterial target. A compound library consisting of 294 members containing metal-binding functional groups was screened against Rickettsia prowazekii MetAP to determine potential inhibitory motifs. The compounds were first screened against the target at a concentration of 10 µM and potential hits were determined to be those exhibiting greater than 50% inhibition of enzymatic activity. These hit compounds were then rescreened against the target in 8-point dose-response curves and 11 compounds were found to inhibit enzymatic activity with IC 50 values of less than 10 µM. Finally, compounds (1-5) were docked against RpMetAP with AutoDock to determine potential binding mechanisms and the results were compared with crystal structures deposited within the PDB., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Methionine aminopeptidase (MetAP) is a class of ubiquitous enzymes essential for the survival of numerous bacterial species. These enzymes are responsible for the cleavage of N-terminal formyl-methionine initiators from nascent proteins to initiate post-translational modifications that are often essential to proper protein function. Thus, inhibition of MetAP activity has been implicated as a novel antibacterial target. We tested this idea in the present study by targeting the MetAP enzyme in the obligate intracellular pathogen Rickettsia prowazekii. We first identified potent RpMetAP inhibitory species by employing an in vitro enzymatic activity assay. The molecular docking program AutoDock was then utilized to compare published crystal structures of inhibited MetAP species to docked poses of RpMetAP. Based on these in silico and in vitro screens, a subset of 17 compounds was tested for inhibition of R. prowazekii growth in a pulmonary vascular endothelial cell (EC) culture infection model system. All compounds were tested over concentration ranges that were determined to be non-toxic to the ECs and 8 of the 17 compounds displayed substantial inhibition of R. prowazekii growth. These data highlight the therapeutic potential for inhibiting RpMetAP as a novel antimicrobial strategy and set the stage for future studies in pre-clinical animal models of infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Louse borne typhus (also called epidemic typhus) was one of man's major scourges, and epidemics of the disease can be reignited when social, economic, or political systems are disrupted. The fear of a bioterrorist attack using the etiologic agent of typhus, Rickettsia prowazekii, was a reality. An attenuated typhus vaccine, R. prowazekii Madrid E strain, was observed to revert to virulence as demonstrated by isolation of the virulent revertant Evir strain from animals which were inoculated with Madrid E strain. The mechanism of the mutation in R. prowazekii that affects the virulence of the vaccine was not known. We sequenced the genome of the virulent revertant Evir strain and compared its genome sequence with the genome sequences of its parental strain, Madrid E. We found that only a single nucleotide in the entire genome was different between the vaccine strain Madrid E and its virulent revertant strain Evir. The mutation is a single nucleotide insertion in the methyltransferase gene (also known as PR028) in the vaccine strain that inactivated the gene. We also confirmed that the vaccine strain E did not cause fever in guinea pigs and the virulent revertant strain Evir caused fever in guinea pigs. We concluded that a single nucleotide insertion in the methyltransferase gene of R. prowazekii attenuated the R. prowazekii vaccine strain E. This suggested that an irreversible insertion or deletion mutation in the methyl transferase gene of R. prowazekii is required for Madrid E to be considered a safe vaccine.

Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Its outer membrane protein B (OmpB) is an immunodominant antigen and plays roles as protective envelope and as adhesins. The observation of the correlation between methylation of lysine residues in rickettsial OmpB and bacterial virulence has suggested the importance of an enzymatic system for the methylation of OmpB. However, no rickettsial lysine methyltransferase has been characterized. Bioinformatic analysis of genomic DNA sequences of Rickettsia identified putative lysine methyltransferases. The genes of the potential methyltransferases were synthesized, cloned, and expressed in Escherichia coli, and expressed proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The methyltransferase activities of the purified proteins were analyzed by methyl incorporation of radioactively labeled S-adenosylmethionine into recombinant fragments of OmpB. Two putative recombinant methyltransferases (rRP789 and rRP027-028) methylated recombinant OmpB fragments. The specific activity of rRP789 is 10- to 30-fold higher than that of rRP027-028. Western blot analysis using specific antibodies against trimethyl lysine showed that both rRP789 and rRP027-028 catalyzed trimethylation of recombinant OmpB fragments. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis showed that rRP789 catalyzed mono-, di-, and trimethylation of lysine, while rRP027-028 catalyzed exclusively trimethylation. To our knowledge, rRP789 and rRP027-028 are the first biochemically characterized lysine methyltransferases of outer membrane proteins from Gram-negative bacteria. The production and characterization of rickettsial lysine methyltransferases provide new tools to investigate the mechanism of methylation of OmpB, effects of methylation on the structure and function of OmpB, and development of methylated OmpB-based diagnostic assays and vaccine candidates.

Here we have characterized the Rickettsia prowazekii RP534 protein, a homologue of the Pseudomonas aeruginosa ExoU phospholipase A (PLA) secreted cytotoxin. Our studies showed that purified recombinant RP534 PLA possessed the predicted PLA(2) and lyso-PLA(2) activities based on what has been published for P. aeruginosa ExoU. RP534 also displayed PLA(1) activity under the conditions tested, whereas ExoU did not. In addition, recombinant RP534 displayed a basal PLA activity that could hydrolyze phosphatidylcholine in the absence of any eukaryotic cofactors. Interestingly, the addition of bovine liver superoxide dismutase 1 (SOD1), a known activator of P. aeruginosa ExoU, resulted in an increased rate of RP534-catalyzed phospholipid hydrolysis, indicating that mechanisms of activation of the ExoU family of PLAs may be evolutionarily conserved. The mechanism of SOD1-dependent stimulation of RP534 was further examined using active site mutants and a fluorogenic phospholipid substrate whose hydrolysis by RP534 over a short time course is measureable only in the presence of SOD1. These studies suggest a mechanism by which SOD1 stimulates RP534 activity once it has bound to the substrate. We also show that antibody raised against RP534 was useful for immunoprecipitating active RP534 from R. prowazekii lysed cell extracts, thus verifying that this protein is expressed and active in rickettsiae isolated from embryonated hen egg yolk sacs., (Copyright © 2011, American Society for Microbiology. All Rights Reserved.)

Rickettsia prowazekii, a parasitic Gram-negative bacterium, is in the second-highest biodefense category of pathogens of the National Institute of Allergy and Infectious Diseases, but only a handful of structures have been deposited in the PDB for this bacterium; to date, all of these have been solved by the SSGCID. Owing to its small genome (about 800 protein-coding genes), it relies on the host for many basic biosynthetic processes, hindering the identification of potential antipathogenic drug targets. However, like many bacteria and plants, its metabolism does depend upon the type II fatty-acid synthesis (FAS) pathway for lipogenesis, whereas the predominant form of fatty-acid biosynthesis in humans is via the type I pathway. Here, the structure of the third enzyme in the FAS pathway, 3-ketoacyl-(acyl-carrier-protein) reductase, is reported at a resolution of 2.25 Å. Its fold is highly similar to those of the existing structures from some well characterized pathogens, such as Mycobacterium tuberculosis and Burkholderia pseudomallei, but differs significantly from the analogous mammalian structure. Hence, drugs known to target the enzymes of pathogenic bacteria may serve as potential leads against Rickettsia, which is responsible for spotted fever and typhus and is found throughout the world.

Rickettsiae are obligate intracellular parasites of eukaryotic cells that are the causative agents responsible for spotted fever and typhus. Their small genome (about 800 protein-coding genes) is highly conserved across species and has been postulated as the ancestor of the mitochondria. No genes that are required for glycolysis are found in the Rickettsia prowazekii or mitochondrial genomes, but a complete set of genes encoding components of the tricarboxylic acid cycle and the respiratory-chain complex is found in both. A 2.4 Å resolution crystal structure of R. prowazekii fumarate hydratase, an enzyme catalyzing the third step of the tricarboxylic acid cycle pathway that ultimately converts phosphoenolpyruvate into succinyl-CoA, has been solved. A structure alignment with human mitochondrial fumarate hydratase highlights the close similarity between R. prowazekii and mitochondrial enzymes.

Rickettsia prowazekii is an obligate intracellular pathogen that possesses a small genome and a highly refined repertoire of biochemical pathways compared to those of free-living bacteria. Here we describe a novel biochemical pathway that relies on rickettsial transport of host cytosolic dihydroxyacetone phosphate (DHAP) and its subsequent conversion to sn-glycerol-3-phosphate (G3P) for synthesis of phospholipids. This rickettsial pathway compensates for the evolutionary loss of rickettsial glycolysis/gluconeogenesis, the typical endogenous source of G3P. One of the components of this pathway is R. prowazekii open reading frame RP442, which is annotated GpsA, a G3P dehydrogenase (G3PDH). Purified recombinant rickettsial GpsA was shown to specifically catalyze the conversion of DHAP to G3P in vitro. The products of the GpsA assay were monitored spectrophotometrically, and the identity of the reaction product was verified by paper chromatography. In addition, heterologous expression of the R. prowazekii gpsA gene functioned to complement an Escherichia coli gpsA mutant. Furthermore, gpsA mRNA was detected in R. prowazekii purified from hen egg yolk sacs, and G3PDH activity was assayable in R. prowazekii lysed-cell extracts. Together, these data strongly suggested that R. prowazekii encodes and synthesizes a functional GpsA enzyme, yet R. prowazekii is unable to synthesize DHAP as a substrate for the GpsA enzymatic reaction. On the basis of the fact that intracellular organisms often avail themselves of resources in the host cell cytosol via the activity of novel carrier-mediated transport systems, we reasoned that R. prowazekii transports DHAP to supply substrate for GpsA. In support of this hypothesis, we show that purified R. prowazekii transported and incorporated DHAP into phospholipids, thus implicating a role for GpsA in vivo as part of a novel rickettsial G3P acquisition pathway for phospholipid biosynthesis.

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. coli 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 was evaluated from the accessibility of hydrophilic, thiol-reactive, methanethiosulfonate reagents to a library of 68 independent cysteine-substitution mutants heterologously expressed in Escherichia coli. The MTS reagents used were MTSES (negatively charged) and MTSET and MTSEA (both positively charged). Mutants F036C, Y042C, and R046C (TM I), K066C and P072C (TM II), and F101C, F105C, F108C, Y113C, and P114C (TM III) had no assayable transport activity, indicating that cysteine substitution at these positions may not be tolerated. All three MTS reagents inhibit the transport of ATP in mutants of TM I (L039C, S043C, S047C, I048C) and TM II (S061C, S063C, T067C, I069C, V070C, A074C). Further, these residues appear to cluster along a single face of the transmembrane domain. Preexposure of MTS-reactive mutants S047C (TM I) and T067C (TM II) to high levels of ATP resulted in protection from MTS-mediated inhibition. This indicated that both TM I and TM II make major contributions to the structure of an aqueous ATP translocation pathway. Finally, on the basis of the lack of accessibility of charged MTS reagents to the thiol groups in mutants of TM III, it appears that TM III is not exposed to the ATP translocation channel. Cysteine substitution of residues constituting a highly conserved "phenylalanine face" in TM III resulted in ablation of ATP transport activity. Further, substituting these phenylalanine residues for either isoleucine or tyrosine also resulted in much lower transport activity, indicating that some property of phenylalanine at these positions that is not shared by cysteine, isoleucine, or tyrosine is critical to translocase activity.

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 was evaluated from the accessibility of three hydrophilic, thiol reactive, methanethiosulfonate reagents to a library of 21 single-cysteine substitution mutants expressed in Escherichia coli. A negatively charged reagent (MTSES) and two positively charged reagents (MTSET and MTSEA) were used. Mutants Q323C and G327C did not tolerate cysteine substitution and were almost completely deficient in ATP transport. The remaining mutants exhibited 25-226% of the cysteine-less parent's transport activity. Five patterns of inhibition of ATP transport by the MTS reagents were observed. (i) ATP transport was not inhibited by any of the three MTS reagents in mutants Q321C, F324C, A332C, and L335C and only marginally in F333C. (ii) Transport activity of mutants F322C, Q326C, and A330C was markedly inhibited by all three reagents. (iii) ATP transport was inhibited by MTSEA in only the largest group of mutants (M334C, I336C, G337C, S338C, N339C, I340C, and I341C). (iv) Transport activity was inhibited by MTSET and MTSEA, whereas high concentrations of MTSES were required to inhibit mutants W328C, V329C, and I331C. However, mutant W328C could be inhibited by MTSES in the presence of sub-K(m) concentrations of the substrate. (v) ATP transport by mutant Y325C was unaffected by MTSEA, but inhibited approximately 50% by MTSET and MTSES. Transport of ATP protected mutants (F322C, W328C, V329C, A330C, and I331C) from MTS inhibition. Mutants in the half of TM VIII that is closest to the cytoplasm were not inhibited well by MTSES or MTSET in either whole cells or inside-out vesicles. 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 (blocked or stimulated) by substrate, and that there is great variation in accessibility to MTS reagents along the length of TM VIII.

To understand further the molecular basis of rickettsial host cell invasion, Rickettsia prowazekii invasion gene homolog (invA) has been characterized. Our previous experiments have shown that InvA is an Ap5A pyrophosphatase, a member of the Nudix hydrolase family, which is up-regulated during the internalization, early growth phase, and exit steps during rickettsial mammalian cell infection. In addition to the molecular characterization, subcellular localization of InvA was investigated. InvA-specific antibodies were raised in mice and used for immunoelectron microscopy. The generated antibodies were shown to recognize InvA and by immunogold labeling showed InvA in the cytoplasm of rickettsiae. A cytoplasmic location for InvA would allow for a rapid response to any internal substance and efficient functioning in hydrolysis of toxic metabolic by-products that are accumulated in the rickettsial cytoplasm during host cell invasion. Protecting bacteria from a hazardous environment could enhance their viability and allow them to remain metabolically active, which is a necessary step for the rickettsial obligate intracellular lifestyle.

The ATP/ADP translocase (Tlc) of Rickettsia prowazekii is a basic protein with isoelectric point (pI)=9.84. It is conceivable, therefore, that basic residues in this protein are involved in electrostatic interactions with negatively charged substrates. We tested this hypothesis by individually mutating all basic residues in Tlc to Cys. Unexpectedly, mutations of only 20 out of 51 basic residues resulted in greater than 80% inhibition of transport activity. Moreover, 12 of 51Cys-substitution mutants exhibited higher than wild-type (WT) activity. At least in one case this up-effect was additive and the double mutant Lys422Cys Lys427Cys transported ATP five-fold better than WT protein. Since in these two single mutants and in the corresponding double mutant K(m)'s were similar to that of WT protein, we conclude that Tlc may have evolved a mechanism that limits the transporter's exchange rate and that at least these two basic residues play a key role in that mechanism. Based on the alignment of 16 Tlc homologs, the loss of activity in the mutants poorly correlates with charge conservation within the Tlc family. Also, despite the presence of three positively charged and one negatively charged intramembrane residues, we have failed to identify potential charge pairs (salt bridges) by either charge reversal or charge neutralization approaches.

The genomic sequence of Rickettsia prowazekii, the obligate intracellular bacterium responsible for epidemic typhus, reveals an uncharacterized invasion gene homolog (invA). The deduced protein of 18,752 Da contains a Nudix signature, the specific motif found in the Nudix hydrolase family. To characterize the function of InvA, the gene was cloned and overexpressed in Escherichia coli. The expressed protein was purified to near homogeneity and subsequently tested for its enzymatic activity against a series of nucleoside diphosphate derivatives. The purified InvA exhibits hydrolytic activity toward dinucleoside oligophosphates (Np(n)N; n > or = 5), a group of cellular signaling molecules. At optimal pH 8.5, the enzyme actively degrades adenosine (5')-pentaphospho-(5')-adenosine into ATP and ADP with a K(m) of 0.1 mM and k(cat) of 1.9 s(-1). Guanosine (5')-pentaphospho-(5')-guanosine and adenosine-(5')-hexaphospho (5')-adenosine are also substrates. Similar to other Nudix hydrolases, InvA requires a divalent metal cation, Mg(2+) or Zn(2+), for optimal activity. These data suggest that the rickettsial invasion protein likely plays a role in controlling the concentration of stress-induced dinucleoside oligophosphates following bacterial invasion.

A new approach to studying membrane topology and permissive sites in membrane proteins expressed in Escherichia coli is described. The method is based on in vitro transposition of mini-Tn5 derivatives bearing dual pho-lac reporters [Alexeyev, M. F., and Winkler, H. H. (1999) J. Mol. Biol. 285, 1503-1513]. Two mini-Tn5 transposons, Tnpholac1 and Tnpholac2, were designed in such a way that their insertions can be converted either by restriction-ligation or by in vivo Cre-lox recombination into either sandwich reporter fusions or short amino acid (aa) tags (25 or 42 aa long). A set of 48 unique insertions in the gene coding for the Rickettsia prowazekii ATP/ADP translocase (Tlc) was generated using Tnpholac2. The topological information generated by these insertions was found in to be in good agreement with the existing topological model. Subsequently, these insertions were converted into both 25 and 42 aa tags, and the activity of the resulting mutants was determined. Also, site-directed mutagenesis was used to construct insertions in the loops, where no transposon hops were discovered. Of 13 extramembrane domains in Tlc, only 3 (loops 7, 10, and 13) were found to be permissive, which is in marked contrast to previous observations in the E. coli lactose permease (LacY), where most insertions in extramembrane domains were demonstrated to be permissive. The permissiveness of the insertion after I368 in TM IX lead us to reconsider the boundaries for this TM by placing I368 on the interface between TM IX and loop 10. Interestingly, the 25 aa insertions consistently have 2-fold higher activity than the corresponding 42 aa insertions, which is also in contrast with observations made on LacY. Finally, in this study we report, for the first time, the frequency of 10 base pair target duplications generated by in vitro Tn5 transposition.

Phospholipase A2 activity by typhus group rickettsiae causes hemolysis in vitro. Rickettsial phospholipase A2 has been proposed to mediate entry into the host cell, escape from the phagosome, and cause injury to host cells by both typhus and spotted fever group rickettsiae. In a rickettsial contact-associated cytotoxicity model, the interaction of Rickettsia prowazekii or R. conorii with Vero cells caused temperature-dependent release of 51Cr from the cells. Treatment of rickettsiae, but not the cells, with a phospholipase A2 inhibitor (bromophenacyl bromide) or with antibody to king cobra venom inhibited cell injury. Rickettsial treatment with bromophenacyl bromide inhibited the release of free fatty acids from the host cell. Neither the inhibitor nor antivenom impaired rickettsial active transport of L-lysine. Thus, host cell injury was mediated by a rickettsial phospholipase A2-dependent mechanism.

The structural gene for DNA polymerase I of Rhizobium leguminosarum was determined. The rhizobium DNA polymerase I consists of 1016 amino acid residues with a calculated molecular weight of 111,491 Dalton. The amino acid sequence comparison with E. coli DNA polymerase I, Thermus aquaticus DNA polymerase I, and Rickettsia prowazekii DNA polymerase I showed that, although 5'-nuclease and DNA polymerase domains are highly conserved, 3' to 5' exonuclease domains are much less conserved. While both R. leguminosarum and R. prowazekii belong to the alpha subdivision of the Proteobacteria on the basis of 16S ribosomal RNA phylogeny, the primary structure of the DNA polymerase I is quite different; the rhizobium DNA polymerase I has 3' to 5' proofreading exonuclease, but the rickettsia DNA polymerase I does not.

Rickettsia prowazekii, the etiological agent of epidemic typhus, is an obligate intracellular bacterium and is apparently unable to synthesize ribonucleotides de novo. Here, we show that as an alternative, isolated, purified R. prowazekii organisms transported exogenous uridyl- and guanylribonucleotides and incorporated these labeled precursors into their RNA in a rifampin-sensitive manner. Transport systems for nucleotides, which we have shown previously and show here are present in rickettsiae, have never been reported in free-living bacteria, and the usual nucleobase and nucleoside transport systems are absent in rickettsiae. There was a clear preference for the monophosphate form of ribonucleotides as the transported substrate. In contrast, rickettsiae did not transport cytidylribonucleotides. The source of rickettsial CTP appears to be the transport of UMP followed by its phosphorylation and the amination of intrarickettsial UTP to CTP by CTP synthetase. A complete schema of nucleotide metabolism in rickettsiae is presented that is based on a combination of biochemical, physiological, and genetic information.

Here, we report the construction and characterization of dual reporters, consisting of both an Escherichia coli alkaline phosphatase (AP) gene and an alpha-fragment of the beta-galactosidase (BG) gene, for studying membrane protein topology by the gene fusion approach. Each of the reporters, when fused to periplasmic domains of polytopic proteins, produces fusions with high AP activity and, when fused to cytoplasmic domains, produces fusions with high BG activity in E. coli strains capable of alpha-complementation. The dual nature of these reporters simplifies interpretation of data obtained with poorly expressed fusions and allows one to evaluate the reliability of topological data. Deleterious effects resulting from the cell's attempt to export the full-length BG are eliminated in this approach. We describe dual indicator plates that allow for discrimination between colonies bearing cytoplasmic fusions, periplasmic fusions, and no fusions. We have generated a set of fusions to the topologically well-studied lactose permease of E. coli and demonstrated that topological information generated by these new reporters is in good agreement with the existing model. We used this new methodology for the determination of membrane topology of the Rickettsia prowazekii ATP/ADP translocase (Tlc). Our results were in agreement with the proposed in silico topological model in which Tlc traverses the cytoplasmic membrane of E. coli 12 times with its N and C termini facing the cytoplasm., (Copyright 1999 Academic Press.)

We have cloned and sequenced the genes encoding cytochrome b (cob) and cytochrome c oxidase subunit I (cox1) from Rickettsia prowazekii, a member of the alpha-proteobacteria. The phylogenetic analysis supports the hypothesis that mitochondria are derived from the alpha-proteobacteria and more specifically from within the Rickettsiaceae. We have estimated that the common ancestor of mitochondria and Rickettsiaceae dates back to more than 1500 million years ago.

Detailed molecular analysis of the Rickettsia prowazekii ATP/ADP translocase, an obligate exchange transport system that is specific for ATP and ADP, has been extremely difficult due to limited quantities of material available from these obligate intracytoplasmic bacteria and by the toxicity and poor expression in recombinant Escherichia coli expression systems. In this study, a stable and controllable system for the increased expression of the rickettsial ATP/ADP translocase was developed in E. coli where the expression of translocase from the bacteriophage T7 promoter in the pET11a vector led to a 26-fold increase in ATP transport activity and a 34-fold increase in translocase protein as compared to the expression with the native rickettsial promoter in E. coli. When compared to R. prowazekii, ATP transport activity was increased sixfold and membrane translocase was increased threefold. Approximately 24% of the translocase protein produced was localized in an inclusion body fraction. This expression system was then used to determine whether the two cysteine residues in the ATP/ADP translocase were essential for activity or expression. The translocase was modified by oligonucleotide-directed site-specific mutagenesis such that the two cysteines were converted to alanines. The ATP transport properties and ATP/ADP translocase production kinetics, translocase protein concentration and subcellular localization were indistinguishable in the wild-type and mutant strains, proving that cysteines play no functional role in the R. prowazekii ATP/ADP translocase and providing a system suitable for cysteine-scanning mutagenesis.

The regulation of the citrate synthase (gltA) and ATP/ADP translocase (tlc) genes of the obligate intracellular bacterium, Rickettsia prowazekii, was analyzed in rickettsia-infected respiration-deficient G14 cells. The level of the gltA mRNAII and the tlc mRNA was much lower in the total RNA isolated from the infected G14 cells grown in 1 g/l glucose (low glucose, GL) medium than in that from infected G14 cells grown in 4.5 g/l glucose (high glucose, GH) medium. However, the level of the gltA mRNAI relative to 16 S rRNA was the same in GL and GH media. An increase in the level of the gltA mRNAII and the tlc mRNA could be observed as early as 2 hrs after shifting from GL to GH medium. We conclude that, under these experimental conditions, the tlc promoter and the gltA promoter P2, but not gltA promoter P1, were transcriptionally regulated.

We have isolated an Arabidopsis thaliana cDNA encoding a highly hydrophobic membrane protein of 589 amino acids which contains 12 potential transmembrane helices and shows a high degree of similarity (43.5% identity, 66.2% similarity) to the ATP/ADP translocase of the Gram-negative bacterium Rickettsia prowazekii, an obligate intracellular parasite responsible for the epidemic typhus. This rickettsial translocator resides in the cytoplasmic membrane and allows the bacterium to exploit the host cytoplasmic ATP pool. We hypothesize that the A. thaliana homolog of the R. prowazekii ATP/ADP translocase is the functional eukaryotic equivalent and resides in the plastid inner envelope membrane where it functions as an ATP importer.

The Rickettsia prowazekii sigma factor was overexpressed, purified, and used to reconstitute RNA polymerase holoenzyme species. R. prowazekii RNA polymerase-promoter complexes were unstable and remained dissociable and heparin sensitive under conditions in which the corresponding Escherichia coli complexes were not. The R. prowazekii core played the major role in determining heparin sensitivity.

The transcripts of the citrate synthase-encoding gene (gltA) in Rickettsia prowazekii (Rp), an obligate intracellular parasitic bacterium, were analyzed by RNase protection (RP), primer extension (PE) and in vitro transcription assays. Analysis of the 5' end of the gltA mRNA by RP and PE assays revealed that there were two gltA mRNAs with the 5' ends located at 16 bp and 307 bp upstream from the gltA coding region. Since these two mRNAs might represent two species of mRNA transcribed from two different promoters or a single transcript that was processed to give two mRNAs, an in vitro transcription analysis with purified Rp RNA polymerase (RNAP) was performed to distinguish these two possibilities. Purified Rp RNAP catalyzed the formation of two transcripts initiated from the same nucleotides indicated by RP and PE. Sequence analysis identified Escherichia coli (Ec) promoter-like sequences immediately upstream from both transcription start points (tsp). The first promoter (promoter P1) had the core sequence TTCTAA-N17-TATACT, was 6 bp upstream from the tsp (base A) and was centered at 37 bp upstream from the coding region. The second promoter (promoter P2) had the core sequence ATGAAA-N17-TAAAGT, was 7 bp upstream from the tsp (base T) and was centered at 329 bp upstream from the coding region. This is the first demonstration of multiple promoters in this obligate intracellular parasite which has implications concerning transcriptional regulation.

The Rickettsia prowazekii (Rp) gyrA gene, which codes for a subunit of DNA gyrase in this obligate intracellular bacterium, has been isolated and characterized. Nucleotide sequence analysis revealed an open reading frame (ORF), initiating with a GTG start codon, of 2718 bp that could encode a protein of 905 amino acids (aa) with a calculated M(r) of 101,048. The Rp gyrase subunit A (GyrA), when compared to GyrA analogs of other bacterial species, exhibited 43 to 50% identity. Alignment of the Rp GyrA aa sequence with the other analogs revealed the presence of a span of additional aa within the putative DNA-binding domain. The lack of an ORF within 865 bp upstream from the Rp gyrA demonstrates a Rp gene organization different from that of characterized gyrA from other species. Despite the similarity to Escherichia coli GyrA, Rp GyrA did not complement an E. coli gyrA temperature-sensitive mutant. However, Rp gyrA was dominant to an E. coli gyrA96 nalidixic-acid-resistant (NalR) mutant, conferring Nal sensitivity when introduced into the NalR E. coli strain.

Southern blot analysis of HindIII-cleaved rickettsial DNA was used for genotypic characterization of the typhus group (TG) species (R. prowazekii, R. typhi, R. canada) and a few species of the spotted fever group (SFG) rickettsiae (R. sibirica, R. conorii, R. akari). Four different DNA probes were employed. PBH11 and PBH13 probes were morphospecific HindIII fragments of R. prowazekii DNA. MW218 probe contained the gene for 51 K antigen and MW264 probe contained the citrate synthase gene of R. prowazekii. All the probes hybridized with the tested TG and SFG rickettsial DNAs, forming from 1 to 5 bands, but they did not with R. tsutsugamushi or C. burnetii DNAs. All the probes demonstrated specific hybridization patterns with TG species and R. akari. PBH11, PBH13 and MW264 probes clearly distinguished R. sibirica and R. conorii from the other tested rickettsiae, but not from each other. However, these two species differed slightly with MW218 probe. Several strains of each species were analyzed in this way and except for strains of R. conorii identical intraspecies patterns were obtained. These data lead us to consider the obtained hybridization patterns as criteria for genotypic identification.

In the obligate intracellular parasitic bacterium, Rickettsia prowazekii, the molar ratio of sigma 73 to core RNA polymerase, that is, the degree of saturation of the core polymerase by the catalytically active sigma factor, was very low. This ratio was determined from the radioactivity in rickettsial RNA polymerase immunoprecipitated from crude extracts of infected L929 cells in which the parasite was exponentially growing. If we assume that, as is true for the sigma subunit, in R. prowazekii and Escherichia coli the beta', and beta subunits of the RNA polymerase have similar methionine and cysteine contents (the radiolabelled amino acids), the molar ratio of sigma 73 to core polymerase in R. prowazekii would be 0.1. This is in striking contrast to E. coli where the ratio is typically 0.4. It remains to be established whether this low sigma saturation results in a limitation of active RNA polymerase in R. prowazekii and contributes to its slow growth.

The gene (sdhA) coding for the flavoprotein subunit (SdhA) of succinate dehydrogenase of the obligate intracellular parasitic bacterium, Rickettsia prowazekii, has been isolated using an oligodeoxyribonucleotide probe to the conserved flavin adenine dinucleotide (FAD)-binding region of characterized flavoproteins. Nucleotide (nt) sequence analysis revealed an open reading frame (ORF) of 1791 bp capable of encoding a protein of 596 amino acids (aa) with a deduced M(r) of 65,444. The deduced aa sequence, when compared to the flavoprotein subunits of Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and Bos taurus, revealed 52.8, 34.0, 65.8 and 52.0% aa identity, respectively. R. prowazekii SdhA produced in E. coli minicells and analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) migrated as a protein of approximately 63 kDa, comparable to the size of the deduced protein. In addition, two proteins of approximately 12 and 41 kDa were also produced in the E. coli minicells. The production of these proteins resulted from additional translational starts within the SdhA coding sequence, suggesting differences between the translational start signals of E. coli and R. prowazekii. Despite the similarity of R. prowazekii SdhA to that of E. coli, the R. prowazekii SdhA did not complement an E. coli sdhA mutant. In addition, analysis of the nt sequence immediately upstream from R. prowazekii sdhA revealed that the rickettsial sdh gene organization differs from that of E. coli and B. subtilis.

In vitro specific transcription by the Rickettsia prowazekii RNA polymerase was investigated. The purified rickettsial RNA polymerase, in striking contrast to that of Escherichia coli, could specifically transcribe two R. prowazekii genes (ATP/ADP translocase and citrate synthase genes) and one E. coli gene (RNA-I) on negative supercoiled plasmids but not the same genes on linear plasmids. Following the specific binding of the rickettsial RNA polymerase to the translocase gene promoter on a linear plasmid, there was no detectable open complex formation. Both the E. coli and the R. prowazekii RNA polymerases worked well when poly(dA-dT).poly(dA-dT) or poly(dI-dC).poly-(dI-dC) was used as template for generalized transcription. However, the rickettsial RNA polymerase, in contrast to the E. coli enzyme, had little activity on poly(dG-dC).poly(dG-dC), a template with a larger number of hydrogen bonds. These data indicate that the rickettsial RNA polymerase is weak, at least relative to E. coli, in the function required for the opening of DNA duplex. It appears that this operation in R. prowazekii is aided by the negative supercoiling and the high 72% AT composition of the rickettsial genome.

The gene (dnaG) coding for DNA primase in the obligate intracellular parasitic bacterium, Rickettsia prowazekii, has been isolated and characterized. An open reading frame (ORF) of 1848 bp capable of encoding 616 amino acids (aa) is located 18 bp upstream from the gene coding for the major sigma factor of R. prowazekii, sigma 73. Based on aa sequence comparisons of DNA primase from R. prowazekii, Escherichia coli, Salmonella typhimurium and Bacillus subtilis, we propose that R. prowazekii dnaG begins 69 bp into the ORF and encodes 593 aa with a calculated M(r) of 68,683. An upstream ORF overlaps 66 of the first 69 bp of the larger R. prowazekii dnaG ORF, suggesting either an overlapping gene structure or the generation of the smaller protein product of 593 aa. Predicted aa sequence of R. prowazekii primase compared to E. coli, S. typhimurium and B. subtilis primases reveals 30.5%, 30.5% and 29.7% aa identity, respectively. The R. prowazekii dnaG gene failed to complement an E. coli dnaG temperature sensitive mutation perhaps due to poor expression of the gene or inability to function properly in E. coli. The gene organization of an ORF followed by DNA primase (dnaG) and then the major sigma factor (rpoD) is consistent with the major macromolecular synthesis operons of E. coli, S. typhimurium and B. subtilis.

The pepA gene, encoding a protein with leucine aminopeptidase activity, was isolated from Rickettsia prowazekii, an obligate intracellular parasitic bacterium. Nucleotide sequence analysis revealed an open reading frame of 1,502 bp that would encode a protein of 499 amino acids with a calculated molecular weight of 53,892, a size comparable to that of the protein produced in Escherichia coli minicells containing the rickettsial gene. Also, heat-stable leucine aminopeptidase activity was demonstrable in an E. coli peptidase-deficient strain containing R. prowazekii pepA. Comparison of the amino acid sequence of the R. prowazekii PepA with the characterized leucine aminopeptidases from E. coli, Arabidopsis thaliana, and bovine eye lens revealed that 39.8, 34.9, and 34.0% of the residues were identical, respectively. Residues proposed to be part of the active site or involved in the binding of metal ions in the bovine metalloenzyme were all conserved in R. prowazekii PepA. However, despite the structural and enzymatic similarity to E. coli PepA, the R. prowazekii protein was unable to complement the cer site-specific, PepA-dependent recombination system found in E. coli that resolves ColE1-type plasmid multimers into their monomeric forms.

The gene coding for the major sigma factor of Rickettsia prowazekii, an obligate intracellular parasitic bacterium, has been isolated utilizing an oligodeoxyribonucleotide as a probe to a conserved region of major sigma factors. Nucleotide sequence analysis revealed an open reading frame of 1905 bp that could encode a protein of 635 amino acids (aa) with a calculated molecular size of 73 kDa (sigma 73). R. prowazekii sigma 73 displayed extensive homology with major sigma factors from a variety of eubacteria. Comparison of the major sigma factors from Escherichia coli and R. prowazekii revealed 44.9% aa identity. R. prowazekii sigma 73 produced in E. coli minicells migrated as a 85-kDa protein when analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. This anomalous migration is characteristic of eubacterial major sigma factors and agrees with the migration noted for the purified rickettsial sigma protein. Despite a similarity to the E. coli sigma 70 encoded by rpoD, R. prowazekii sigma 73 did not complement E. coli rpoD temperature-sensitive mutants.

Thymidylate biosynthesis via the methylation of dUMP is required for DNA replication in Rickettsia prowazekii, an obligate intracytoplasmic bacterium. In theory, dUMP synthesis could occur either by the deamination of deoxycytidine nucleotides or by the reduction of uridine nucleotides. Accordingly, the incorporation of both radiolabeled cytidine and uridine into the thymidylate of R. prowazekii was examined. After DNA hydrolysis and high-performance liquid chromatography, it was determined that 85% of the rickettsial thymidylate was derived from cytidine and the remaining 15% was derived from uridine. These findings were supported by the identification of a dCTP deaminase activity in extracts of R. prowazekii. Extracts of R. prowazekii deaminated 1.7 +/- 0.3 nmol of dCTP/min/mg of protein (a value calculated to suffice for rickettsial growth), and no measurable activity was observed with dCMP as the substrate.

Classical genetic approaches useful with free-living bacteria are difficult to apply to the rickettsiae. Although rickettsial mutants have been isolated over the years, the genetic basis of these mutants is unknown, limiting their usefulness. The application of molecular biological techniques to rickettsial studies has provided the opportunity to isolate and study specific genes. Genes encoding metabolic enzymes from rickettsiae were cloned in Escherichia coli and shown to retain their regulatory properties, suggesting that recombinant DNA technology may be useful for studies of rickettsial enzymes and regulatory mechanisms. The potential use of rickettsial surface components, or virulence factors as possible antigens for protective subunit vaccines, has led to the cloning and expression in E. coli, of rickettsial chromosomal and plasmid genes encoding outer membrane proteins. The number of genes characterized in recent years has increased dramatically giving rise to an increasing source of information on rickettsial gene structure. Plasmids have only been identified in C. burnetii and possibly Rochalimaea quintana. The plasmid sequences present in C. burnetii are highly conserved suggesting that they are important to the growth and virulence of this organism. To understand the role of genes in the rickettsia-host relationship, it is critical that a genetic exchange system be developed. The recent description of transformation of R. quintana by electroporation is an important first step in this direction. The ability to introduce genetic material is necessary to address questions that cannot be resolved by studying rickettsial gene expression in E. coli.

Our understanding of the biology of the rickettsiae, organisms that are the archetype of the obligate intracytoplasmic bacterial parasites, remains muddy and fragmentary. For example although we all appreciate that the rickettsiae can exploit their unique environment, the host cell cytoplasm, but are unable to grow axenically, the basis of this fact is still one of microbiology's central mysteries. It is unfortunate, but true, that because of the inherent difficulty of working within this system, progress on the answers to such questions will be slow and laborious. However, with the application of molecular biological methods, that is, the powerful modern approaches of genetics and biochemistry, the rickettsiology community has the realistic prospect that this field is far from being at a stand-still and that significant increases in our comprehension of the fundamental problems of rickettsial biology are occurring and will continue to occur at ever accelerating rates. Some examples, both in terms of scientific conclusions and technical approaches, of the progress made in recent years and expectations for the near future will be presented.

DNA sequences from specific genes, amplified by the polymerase chain reaction technique, were used as substrata for nonisotopic restriction endonuclease fragment length polymorphism differentiation of rickettsial species and genotypes. The products amplified using a single pair of oligonucleotide primers (derived from a rickettsial citrate synthase gene sequence) and cleaved with restriction endonucleases were used to differentiate almost all recognized species of rickettsiae. A second set of primers was used for differentiation of all recognized species of closely related spotted fever group rickettsiae. The procedure circumvents many technical obstacles previously associated with identification of rickettsial species. Multiple amplified DNA digest patterns were used to estimate the intraspecies nucleotide sequence divergence for the genes coding for rickettsial citrate synthase and a large antigen-coding gene of the spotted fever group rickettsiae. The estimated relationships deduced from these genotypic data correlate reasonably well with established rickettsial taxonomic schemes.

Cultured human umbilical vein-derived endothelial cells were incubated with typhus rickettsiae, and supernatants were examined for the presence of prostaglandins I2 (PGI2) and E2 (PGE2). Cells incubated with metabolically active rickettsiae secreted significantly more PGI2 and PGE2 than did those incubated with buffer alone or with killed rickettsiae. The amount of PGI2 secreted was directly related to the number of hemolytically active rickettsiae present; abolishing rickettsial hemolytic activity abolished their effect on PGI2 secretion. Mice injected with a lethal dose of native typhus rickettsiae exhibited a rapid rise in circulating PGI2 levels; mice given hemolytically inactive rickettsiae survived and exhibited no rise in plasma PGI2 levels. Finally, endothelial cells were infected with rickettsiae, and secretion of prostaglandins was monitored during rickettsial multiplication; intracellular accumulation of rickettsiae resulted in endothelial destruction and a dramatic increase in endothelial secretion of PGI2 and PGE2. Therefore, typhus rickettsiae can increase endothelial secretion of arachidonate-derived autocoids.

The DNA-dependent RNA polymerase was purified from Rickettsia prowazekii, an obligate intracellular bacterial parasite. Because of limitation of available rickettsiae, the classical methods for isolation of the enzyme from other procaryotes were modified to purify RNA polymerase from small quantities of cells (25 mg of protein). The subunit composition of the rickettsial RNA polymerase was typical of a eubacterial RNA polymerase. R. prowazekii had beta' (148,000 daltons), beta (142,000 daltons), sigma (85,000 daltons), and alpha (34,500 daltons) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The appropriate subunits of the rickettsial RNA polymerase bound to polyclonal antisera against Escherichia coli core polymerase and E. coli sigma 70 subunit in Western blots (immunoblots). The enzyme activity was dependent on all four ribonucleoside triphosphates, Mg2+, and a DNA template. Optimal activity occurred in the presence of 10 mM MgCl2 and 50 mM NaCl. Interestingly, in striking contrast to E. coli, approximately 74% of the rickettsial RNA polymerase activity was associated with the rickettsial cell membrane at a low salt concentration (50 mM NaCl) and dissociated from the membrane at a high salt concentration (600 mM NaCl).

Cosmid clone banks of Rickettsia prowazekii genomic DNA were established in Escherichia coli and screened for expression of the rickettsial carrier-mediated ADP/ATP translocator. Out of 2700 clones screened, a single clone, designated MOB286, accumulated radioactivity when incubated with [alpha-32P]ATP in 100 mM sodium phosphate buffer. This clone carried a plasmid, pMW286, containing a 9-kilobase-pair insert of rickettsial DNA, as established by DNA X DNA hybridizations. Transformation studies with purified pMW286 established that the ability of E. coli cells to accumulate radioactivity was mediated by the recombinant plasmid. Results from experiments in which [3H]ATP was substituted for [alpha-32P]ATP strongly suggested that the radiolabeled ATP was transported intact. Furthermore, [3H]ATP was incorporated into 10% (wt/vol) trichloroacetic acid-precipitable material in a time-dependent manner. Uptake of ATP was also temperature-dependent, insensitive to atractyloside, N-ethylmaleimide, and dinitrophenol, and specific for ADP and ATP. Efflux of radiolabeled nucleotide was observed in the presence of extracellular ADP or ATP but not AMP and was not observed in the absence of extracellular adenine nucleotides. The successful cloning and expression of the rickettsial ADP/ATP translocator in E. coli will permit better characterization of rickettsial bioenergetics and of the metabolic regulation of obligate intracellular parasitism.

Purified Rickettsia prowazekii cells were able to transport L-proline. The influx of this amino acid had a Kt of 14 microM and a Vmax of about 64 pmol/min per mg of protein. Proline could not be transported by heat-killed or metabolically poisoned rickettsiae or at 0 degrees C. The uptake of proline was linear for almost 2 h. More than 90% of the accumulated intracellular radioactivity was proline. This intracellular pool could not be chased out of the cell by excess non-radioactive proline and did not exit into a proline-free medium. These results indicate that intracellular proline was bound or that the cell had a very limited efflux component for proline transport. The influx of proline was specific: among various analogs tested, only 3,4-dehydro-D,L-proline was effective in inhibiting proline uptake. R. prowazekii cells were unable to utilize proline as an energy source to drive hemolysis, and no measurable evolution from the rickettsiae of CO2 derived from proline occurred. The activities of the enzymes pyrroline-5-carboxylate-reductase and pyrroline-5-carboxylate dehydrogenase were not detectable. These enzymes are important in anabolism and catabolism of proline, respectively, and, if present in R. prowazekii have activities less than 1% of those in Escherichia coli.

The interaction of large numbers of viable Rickettsia prowazekii cells with L-cells results in the expression of a phospholipase A activity with the concomitant release of free fatty acids and lysophosphatides from the phospholipids of the L-cell. About 50% of rickettsiae present in the suspension that was centrifuged onto an L-cell monolayer at 0 degree C to effect this interaction formed a tight L-cell-rickettsiae association from which the rickettsiae could not be removed by simple washing. Both the L-cell-rickettsiae association and the rickettsiae before association with L-cells interact with N-ethylmaleimide, so that the subsequent expression of the phospholipase A activity was inhibited (treatment of the L-cells with N-ethylmaleimide before centrifugation does not inhibit phospholipase activity). However, treatment of this association with 2,4-dinitrophenol and KCN caused much less inhibition of this phospholipase A activity than did treatment of the rickettsiae with these agents before centrifugation onto the L-cells. Incubation of the L-cell-rickettsiae association for a short time at 35 degrees C resulted in a very low level of free fatty acid formation and changed this association to an activated complex in which the phospholipase A activity was no longer sensitive to the inhibitory effects of N-ethylmaleimide. The characteristics of the association and activated complex were stable: after a 2-h incubation at 0 degrees C, the association and the activated complex retained both their basal phospholipase A activities and their characteristic responses to N-ethylmaleimide treatment. In scanning electron micrographs of the activated complexes, the rickettsiae that were initially attached were no longer visible after 45 min at 35 degrees C, and the surface of the L-cell appeared to have been etched away. These activated complexes provide a system in which modulators of the phospholipase A can be investigated without the confusion caused by the first-step receptor interaction between rickettsiae and their host cells.

Six strains of Rickettsia prowazekii, two derived from human infections and four isolated from flying squirrels, two strains of R. typhi, and the single available strain of R. canada, were characterized by several biochemical procedures. The electrophoretic patterns on polyacrylamide gels of rickettsial proteins solubilized by sodium dodecyl sulfate revealed several species differences, but strains of the same species appeared to have identical patterns. Cytoplasmic fractions of the rickettsiae were examined for enzymatic activities and for polyacrylamide gel isoelectric focusing patterns. Some species differences were encountered in the activities or ratios of activities of glutamate-oxaloacetate transaminase, glutamate dehydrogenase, and malate dehydrogenase. When polyacrylamide gels were stained for malate dehydrogenase after electrophoresis, a single band became apparent with single extracts or mixtures of two strains of R. prowazekii, but two bands were seen with mixtures of a strain of R. prowazekii and one of R. typhi. The isoelectric focusing patterns of the soluble proteins revealed numerous species differences, especially between R. canada and the other two species, and a few differences among the strains of R. prowazekii. The patterns of the two human strains, Breinl and E(R), differed in at least one location, and both differed from the flying squirrel strains in the displacement of one band. One of the flying squirrel strains, GvF-16, contained a protein band not seen in the other five strains. Despite these minor differences, a striking similarity was revealed by all the biochemical tests performed between the R. prowazekii strains of human and flying squirrel origin.

Incubation of Rickettsia prowazeki with sheep or human erythrocytes resulted in lysis of the erythrocytes and formation of free fatty acids and lysophosphatides. Inhibitors of lysis were also invariably inhibitors of this phospholipase A activity. The target for this activity was the glycerophospholipids of the erythrocyte and not those of the rickettsia. Rickettsial phosphatidylethanolamine labeled with (32)PO(4) or [(3)H]acetate remained intact during lysis, and the composition of the free fatty acids released resembled that of the erythrocyte species used and not the rickettsiae. The products of hydrolysis remained associated with the sedimentable material in the reaction mixture under the usual conditions but partitioned into the supernatant fluid when bovine serum albumin was present. Initially, the time course of phospholipase A activity and lysis was identical, but the release of free fatty acids continued for a short time after the release of hemoglobin was complete. Both the inner and outer leaflets of the erythrocyte membrane were accessible to this rickettsial phospholipase A activity since both phosphatidylcholine and phosphatidylethanolamine were substrates in human erythrocytes. The questions of whether rickettsiae possess their own phospholipase A or activate a latent erythrocyte enzyme and what the role of the energy requirement is in these processes remain unanswered.

The Rickettsia prowazekii citrate synthase (gltA) gene, previously cloned in Escherichia coli, was localized to a 2.0-kilobase chromosomal fragment. DNA sequence analysis of a portion of this fragment revealed an open reading frame of 1,308 base pairs that encodes a protein of 435 amino acids with a molecular weight of 49,171. This translation product is comparable in size to both the E. coli and pig heart citrate synthase monomers and to the protein synthesized in E. coli minicells containing the rickettsial gene. Comparisons between the deduced amino acid sequence of R. prowazekii citrate synthase and those of the E. coli and pig heart enzymes revealed extensive homology (59%) between the two bacterial proteins. In contrast, only 20% of the rickettsial enzyme residues were shared with the functionally similar pig heart enzyme residues. Upstream from the open reading frame and in close proximity to one another, sequences with homology to E. coli consensus sequences for RNA polymerase and ribosome binding were identified. S1 nuclease mapping experiments demonstrated that the start of transcription for this gene in E. coli was located in the upstream region. Codon usage in the rickettsial gltA gene was found to be very biased and differed from the pattern observed in E. coli. Adenine and uracil were used preferentially in the third base position of rickettsial codons.

Citrate synthase [citrate (si)-synthase] (EC 4.1.3.7) was partially purified from extracts of highly purified typhus rickettsiae (Rickettsia prowazekii). Molecular exclusion and affinity column chromatography were used to prepare 200-fold-purified citrate synthase that contained no detectable malate dehydrogenase (EC 1.1.1.37) activity. Rickettsial malate dehydrogenase also was partially purified (200-fold) via this purification procedure. Catalytically active citrate synthase exhibited a relative molecular weight of approximately 62,000 after elution from a calibrated Sephacryl S-200 column. Acetyl coenzyme A saturation of partially purified enzyme was sensitive to strong competitive inhibition with adenylates (ATP greater than ADP much greater than AMP). [beta,gamma-methylene]ATP, dATP, and dADP also caused strong inhibition, but guanosine and cytosine nucleotides were significantly less inhibitory. Adenylates had no effect on oxalacetate saturation kinetics when acetyl coenzyme A was present in high concentration (greater than or equal to 50 microM). Neither NADH nor alpha-ketoglutarate affected the saturation kinetics of rickettsial citrate synthase. Thus, citrate synthase from R. prowazekii exhibits greater similarity to the eucaryotic and gram-positive procaryotic enzymes than to citrate synthase from free-living gram-negative bacteria. These results represent the first characterization of a highly purified key regulatory enzyme from these obligate intracellular parasitic bacteria.