Complete genome sequence of Rickettsia typhi and comparison with sequences of other rickettsiae

Rickettsia typhi, the causative agent of murine typhus, is an obligate intracellular bacterium with a life cycle involving both vertebrate and invertebrate hosts. Here we present the complete genome sequence of R. typhi (1,111,496 bp) and compare it to the two published rickettsial genome sequences: R. prowazekii and R. conorii. We identified 877 genes in R. typhi encoding 3 rRNAs, 33 tRNAs, 3 noncoding RNAs, and 838 proteins, 3 of which are frameshifts. In addition, we discovered more than 40 pseudogenes, including the entire cytochrome c oxidase system. The three rickettsial genomes share 775 genes: 23 are found only in R. prowazekii and R. typhi, 15 are found only in R. conorii and R. typhi, and 24 are unique to R. typhi. Although most of the genes are colinear, there is a 35-kb inversion in gene order, which is close to the replication terminus, in R. typhi, compared to R. prowazekii and R. conorii. In addition, we found a 124-kb R. typhi-specific inversion, starting 19 kb from the origin of replication, compared to R. prowazekii and R. conorii. Inversions in this region are also seen in the unpublished genome sequences of R. sibirica and R. rickettsii, indicating that this region is a hot spot for rearrangements. Genome comparisons also revealed a 12-kb insertion in the R. prowazekii genome, relative to R. typhi and R. conorii, which appears to have occurred after the typhus (R. prowazekii and R. typhi) and spotted fever (R. conorii) groups diverged. The three-way comparison allowed further in silico analysis of the SpoT split genes, leading us to propose that the stringent response system is still functional in these rickettsiae.     

S-adenosylmethionine transport in Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells. Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm. Analysis of the R. prowazekii Madrid E genome sequence revealed the presence of a mutation in the rickettsial metK gene, the gene encoding the enzyme responsible for the synthesis of S-adenosylmethionine (AdoMet). Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter. We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified. The influx of AdoMet into rickettsiae was a saturable process with a K(T) of 2.3 micro M. Transport was inhibited by S-adenosylethionine and S-adenosylhomocysteine but not by sinfungin or methionine. Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by N-ethylmaleimide. AdoMet transporters with similar properties were also identified in the Breinl strain of R. prowazekii and in Rickettsia typhi. By screening Escherichia coli clone banks for AdoMet transport, the R. prowazekii gene coding for a transporter, RP076 (sam), was identified. AdoMet transport in E. coli containing the R. prowazekii sam gene exhibited kinetics similar to that seen in rickettsiae. The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.     

Seroimmunologic monitoring of microorganisms of Rickettsia and Bartonella species microorganisms in the Moscow region

Serological study of 788 blood sera, taken from residents of the Moscow region was conducted using antigens of microorganisms of the genera Rickettsia and Bartonella. The first group under examination consisted of 355 patients with diagnosed diseases of nonreckettsial nature. The second group includes 433 healthy adults working at a meat processing and packing factory. The main method used for sera survey was the indirect immunofluorescence test. In the sera taken from the first group of subjects specific antibodies to R. prowazekii, R. typhi, B. quintana, B. henselae antigens were detected in 2.3%, 5.1%, 4.0% and 2.9% of serum samples respectively. In the serum samples taken from the second group the proportion of antibodies to R. prowazekii, R. typhi, B. quintana, B. henselae antigens was different: 0.5%, 3.3%, 1.7% and 4.0% respectively. In total, specific antibodies to R. typhi and B. henselae prevailed over specific antibodies to R. prowazekii and B. quintana twofold.     

Development of a physical map for three Frankia strains and a partial genetic map for Frankia EuI1c

Physical maps for Frankia strains CcI3, EAN1pec and EuI1c chromosomes were constructed by the use of macrorestriction analysis and pulsed-field gel electrophoresis (PFGE). The restriction enzymes Ase I, Pme I, Swa I and Ssp I were used to cut the Frankia chromosome into a limited number of large fragments and for double digestions. The genomes sizes, as determined by the addition of the estimated fragment sizes, were 5430 ± 35 kb, 9101 ± 109 kb and 8105 ± 842 kb for strains CcI3, EAN1pec and EuI1c, respectively. A complete physical map was achieved by the analysis of PFGE for the single and double digestions and by two-dimensional PFGE to determine doublets and overlapping fragments. For strain EuI1c, a partial genetic map was also constructed by positioning the 16S rRNA, gln II, gln A and hbo O genes on the physical map. PFGE analysis of DNA with and without proteinase K treatment together with the other results suggested a circular genome.     

Use of quantitative measures of gene order similarity to phylogenetic reconstructions (exemplified by bacteria of the genus Rickettsia)

Data reflecting evolutionary changes in chromosomal gene order can be used for phylogenetic reconstructions along with the results of nucleotide sequence comparison. By the example of bacteria of the genus Rickettsia, we have shown that phylogenetic reconstructions based on quantitative estimates of the similarity and cladistic analysis of gene order data, may, in some cases, amend and fill up classical phylogenetic trees. When applied, these approaches enabled us to substantiate the hypothesis that Rickettsia felis species had split before the typhus (R. typhi, R. prowazekii) and spotted fever (R. connorii) group divergence and thus R. felis does not belong to the latter group. In general, rickettsias evolved towards increasing intracellular parasitic specialization. Five Rickettsia species whose genomes have been sequenced and annotated completely actually form an evolutionary series R. hellii-R. felis-R. connorii-R. prowazekii-R. typhi. Within this series, a reduction in genome size and rapid decrease of genome rearrangement rates (genome plasticity loss) gradually occur.     

Mariner-based transposon mutagenesis of Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile mariner-based, Himar1 transposon system to generate insertional mutants of R. prowazekii. A transposon containing the R. prowazekii arr-2 rifampin resistance gene and a gene coding for a green fluorescent protein (GFP(UV)) was constructed and placed on a plasmid expressing the Himar1 transposase. Electroporation of this plasmid into R. prowazekii resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques, R. prowazekii transformants expressing GFP(UV) were easily visible by fluorescence microscopy. Thus, a mariner-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFP(UV) fluorescence.     

The surface antigens of Rickettsia prowazekii studied with monoclonal antibodies]

R. prowazekii antigens have been tested with the use of monoclonal antibodies (McAb) to different epitopes of the microorganism. As revealed in these tests, McAb B4/4 and A-3/D, active against species-specific thermolabile antigen, interact with protein having a molecular weight of 90-120 KD. McAb C5/2, active against thermostable group antigen common with that of Rickettsia typhi, interact with LPS-like antigen having a molecular weight of 30 KD. Ultrastructural immunochemical studies have revealed that both R. prowazekii antigens are located on surface structures of rickettsiae, such as the microcapsule and cell wall.     

Establishment of a replicating plasmid in Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, grows only within the cytosol of eukaryotic host cells. This obligate intracellular lifestyle has restricted the genetic analysis of this pathogen and critical tools, such as replicating plasmid vectors, have not been developed for this species. Although replicating plasmids have not been reported in R. prowazekii, the existence of well-characterized plasmids in several less pathogenic rickettsial species provides an opportunity to expand the genetic systems available for the study of this human pathogen. Competent R. prowazekii were transformed with pRAM18dRGA, a 10.3 kb vector derived from pRAM18 of R. amblyommii. A plasmid-containing population of R. prowazekii was obtained following growth under antibiotic selection, and the rickettsial plasmid was maintained extrachromosomally throughout multiple passages. The transformant population exhibited a generation time comparable to that of the wild type strain with a copy number of approximately 1 plasmid per rickettsia. These results demonstrate for the first time that a plasmid can be maintained in R. prowazekii, providing an important genetic tool for the study of this obligate intracellular pathogen.     

Pseudogenes, junk DNA, and the dynamics of Rickettsia genomes

Studies of neutrally evolving sequences suggest that differences in eukaryotic genome sizes result from different rates of DNA loss. However, very few pseudogenes have been identified in microbial species, and the processes whereby genes and genomes deteriorate in bacteria remain largely unresolved. The typhus-causing agent, Rickettsia prowazekii, is exceptional in that as much as 24% of its 1.1-Mb genome consists of noncoding DNA and pseudogenes. To test the hypothesis that the noncoding DNA in the R. prowazekii genome represents degraded remnants of ancestral genes, we systematically examined all of the identified pseudogenes and their flanking sequences in three additional Rickettsia species. Consistent with the hypothesis, we observe sequence similarities between genes and pseudogenes in one species and intergenic DNA in another species. We show that the frequencies and average sizes of deletions are larger than insertions in neutrally evolving pseudogene sequences. Our results suggest that inactivated genetic material in the Rickettsia genomes deteriorates spontaneously due to a mutation bias for deletions and that the noncoding sequences represent DNA in the final stages of this degenerative process.     

Copy number of the 16S rRNA gene in Rickettsia prowazekii.

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.     

Obligate intracellular parasites: Rickettsia prowazekii and Chlamydia trachomatis.

Transitions to obligate intracellular parasitism have occurred at numerous times in the evolutionary past. The genome sequences of two obligate intracellular parasites, Rickettsia prowazekii and Chlamydia trachomatis, were published last year. A comparative analysis of these two genomes has revealed examples of reductive convergent evolution, such as a massive loss of genes involved in biosynthetic functions. In addition, both genomes were found to encode transport systems for ATP and ADP, not otherwise found in bacteria. Here, we discuss adaptations to intracellular habitats by comparing the information obtained from the recently published genome sequences of R. prowazekii and C. trachomatis.     

Monoclonal antibodies to the species-specific and group antigens of Rickettsia prowazekii]

A number of hybridomas to different R. prowazekii determinants were obtained by the hybridization of spleen cells of BALB/c mice immunized with R. prowazekii corpuscular and soluble antigens. Some of the monoclonal antibodies (McAb) reacted with R. prowazekii thermolabile species-specific protein and did not react with R. typhi antigens (McAb of batches B4/4 and A-D3). McAb C5/2 and A3/2 reacted with the group thermostable antigen, common for R. prowazekii and R. typhi. McAb to the species-specific thermolabile antigen belonged to IgG2a. The McAb thus obtained permit the identification of R. prowazekii and R. typhi and the solution of the problem of the intragroup differentiation of rickettsiae belonging to the typhus group.     

Transposon mutagenesis of the obligate intracellular pathogen Rickettsia prowazekii

Genetic analysis of Rickettsia prowazekii has been hindered by the lack of selectable markers and efficient mechanisms for generating rickettsial gene knockouts. We have addressed these problems by adapting a gene that codes for rifampin resistance for expression in R. prowazekii and by incorporating this selection into a transposon mutagenesis system suitable for generating rickettsial gene knockouts. The arr-2 gene codes for an enzyme that ADP-ribosylates rifampin, thereby destroying its antibacterial activity. Based on the published sequence, this gene was synthesized by PCR with overlapping primers that contained rickettsial codon usage base changes. This R. prowazekii-adapted arr-2 gene (Rparr-2) was placed downstream of the strong rickettsial rpsL promoter (rpsL(P)), and the entire construct was inserted into the Epicentre EZ::TN transposome system. A purified transposon containing rpsL(P)-Rparr-2 was combined with transposase, and the resulting DNA-protein complex (transposome) was electroporated into competent rickettsiae. Following selection with rifampin, rickettsiae with transposon insertions in the genome were identified by PCR and Southern blotting and the insertion sites were determined by rescue cloning and inverse PCR. Multiple insertions into widely spaced areas of the R. prowazekii genome were identified. Three insertions were identified within gene coding sequences. Transposomes provide a mechanism for generating random insertional mutations in R. prowazekii, thereby identifying nonessential rickettsial genes.     

Deamination of deoxycytidine nucleotides by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

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.     

Instability of Rickettsia prowazekii RNA polymerase-promoter complexes.

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.     

Reduction of ribonucleotides by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

Rickettsia prowazekii, an obligate intracellular parasitic bacterium, was shown to have a ribonucleotide reductase that would allow the rickettsiae to obtain the deoxyribonucleotides needed for DNA synthesis from rickettsial ribonucleotides rather than from transport. In the presence of hydroxyurea, R. prowazekii failed to grow in mouse L929 cells or SC2 cells (a hydroxyurea-resistant cell line), which suggested that R. prowazekii contains a functional ribonucleotide reductase. This enzymatic activity was demonstrated by the conversion of ADP to dADP and CDP to dCDP, using (i) a crude extract of Renografin-purified R. prowazekii that had been harvested from infected yolk sacs and (ii) high-performance liquid chromatographic analysis. The rickettsial ribonucleotide reductase utilized ribonucleoside diphosphates as substrates, required magnesium and a reducing agent, and was inhibited by hydroxyurea. ADP reduction was stimulated by dGTP and inhibited by dATP. CDP reduction was stimulated by ATP and adenylylimido-diphosphate and inhibited by dATP and dGTP. These characteristics provided strong evidence that the rickettsial enzyme is a nonheme iron-containing enzyme similar to those found in mammalian cells and aerobic Escherichia coli.     

Conserved gene cluster at replication origins of the alpha-proteobacteria Caulobacter crescentus and Rickettsia prowazekii

A 30-kb region surrounding the replication origin in Caulobacter crescentus was analyzed. Comparison to the genome sequence of another alpha-proteobacterium, Rickettsia prowazekii, revealed a conserved cluster of genes (RP001, hemE, hemH, and RP883) that overlaps the established origin of replication in C. crescentus and the putative origin of replication in R. prowazekii. The genes flanking this cluster differ between these two organisms. We therefore propose that this conserved gene cluster can be used to identify the origin of replication in other alpha-proteobacteria.     

Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes

Abstract The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases. The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb. The Not I and S fi I digestion patterns of the genomes of several V. cholerae straimns belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP). RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V. cholerae .