Herbert H. Winkler

The genome sequence of Rickettsia prowazekii and the origin of mitochondria.

We describe here the complete genome sequence (1,111,523 base pairs) of the obligate intracellular parasite Rickettsia prowazekii, the causative agent of epidemic typhus. This genome contains 834 protein-coding genes. The functional profiles of these genes show similarities to those of mitochondrial genes: no genes required for anaerobic glycolysis are found in either R. 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 R. prowazekii. In effect, ATP production in Rickettsia is the same as that in mitochondria. Many genes involved in the biosynthesis and regulation of biosynthesis of amino acids and nucleosides in free-living bacteria are absent from R. prowazekii and mitochondria. Such genes seem to have been replaced by homologues in the nuclear (host) genome. The R. prowazekii genome contains the highest proportion of non-coding DNA (24%) detected so far in a microbial genome. Such non-coding sequences may be degraded remnants of ‘neutralized’ genes that await elimination from the genome. Phylogenetic analyses indicate that R. prowazekii is more closely related to mitochondria than is any other microbe studied so far.

Non-mitochondrial ATP transport

Exchange of organelle ATP with cytosolic ADP through the ADP/ATP carrier is a well-characterized feature of mitochondrial metabolism. Obligate intracellular bacteria, such as Rickettsia prowazekii, and higher-plant plastids possess another type of adenylate transporter, which exchanges bacterial or plastidic ADP for ATP from the eukaryotic (host cell) cytoplasm. The bacterial and plastidic transporters are similar but do not share significant sequence similarities with the mitochondrial carrier. Recent molecular and biochemical studies are providing deeper insight into the functional and evolutionary relationships between the bacterial and the plant transport proteins.

Phylogenetic relationships of non-mitochondrial nucleotide transport proteins in bacteria and eukaryotes

Current knowledge about the nucleotide metabolism of intracellular bacteria is very limited. Here we report on the identification of nucleotide transport proteins (NTT) of two obligate endoparasites, Caedibacter caryophila and Holospora obtusa, both alpha-proteobacteria, which reside in the vegetative macronucleus of Paramecium caudatum. For comparative studies, we also identified the first nucleotide transporter in chloroplasts of a red alga, i.e. Galdieria sulphuraria, and further homologs in plant chloroplasts. Heterologous expression of the NTT proteins from C. caryophila, H. obtusa, and G. sulphuraria in Escherichia coli demonstrate that the nucleotide influx mediated by these transporters is specific for ATP and ADP. The NTT proteins of C. caryophila and H. obtusa exhibit substantial sequence identity with their counterparts in chloroplasts and intracellular bacterial pathogens of humans, but not with the nucleotide transport system of mitochondria. Comprehensive phylogenetic analyses of bacterial and chloroplast NTT proteins showed that homologs in chloroplasts from plants, and green, red, stramenopile and glaucocystophyte algae are monophyletic. In contrast, the evolutionary relationships of the bacterial counterparts appear highly complex. In the presented phylogeny, NTT proteins of C. caryophila and H. obtusa are only distantly related to one another, although these two taxa are close relatives in 16S rRNA trees. The tree topology indicates that some bacterial NTT paralogs have arisen by gene duplications and others by horizontal transfer.

Properties of the Glucose-6-Phosphate Transporter from Chlamydia pneumoniae (HPTcp) and the Glucose-6-Phosphate Sensor from Escherichia coli (UhpC)

The amino acid sequence of the proposed glucose-6-phosphate (Glc6P) transporter from Chlamydia pneumoniae (HPTcp; hexose phosphate transporter [Chlamydia pneumoniae]) exhibits a higher degree of similarity to the Escherichia coli Glc6P sensor (UhpC) than to the E. coli Glc6P transporter (UhpT). Overexpression of His-UhpC in a UhpT-deficient E. coli strain revealed that the sensor protein is also able to transport Glc6P and exhibits an apparent K(m) ((Glc6P)) of 25 microM, whereas His-HPTcp exhibits an apparent K(m)( (Glc6P)) of 98 microM. His-HPTcp showed a four-times-lower specific activity than His-UhpT but a 56-times-higher specific activity than His-UhpC. Like His-UhpT and His-UhpC, the carrier His-HPTcp performs a sugar-phosphate/inorganic-phosphate antiporter mode of transport. Surprisingly, while physiological concentrations of inorganic phosphate competitively inhibited transport mediated by the E. coli proteins His-UhpT and His-UhpC, transport mediated by His-HPTcp was not inhibited. Interestingly, C(3)-organophosphates stimulated His-HPTcp activity but not His-UhpT- or His-UhpC-catalyzed Glc6P transport. In contrast to His-UhpC, the His-HPTcp protein does not act as a Glc6P sensor in the uhp regulon.

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 cells 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.

Transformation of Rickettsia prowazekii to Erythromycin Resistance Encoded by the Escherichia coli ereB Gene

Rickettsia prowazekii, the etiologic agent of epidemic typhus, is an obligate, intracytoplasmic, parasitic bacterium. Recently, the transformation of this bacterium via electroporation has been reported. However, in these studies identification of transformants was dependent upon either selection of an R. prowazekii rpoB chromosomal mutation imparting rifampin resistance or expression of the green fluorescent protein and flow cytometric analysis. In this paper we describe the expression in R. prowazekii of the Escherichia coli ereB gene. This gene codes for an erythromycin esterase that cleaves erythromycin. To the best of our knowledge, this is the first report of the expression of a nonrickettsial, antibiotic-selectable gene in R. prowazekii. The availability of a positive selection for rickettsial transformants is an important step in the characterization of genetic analysis systems in the rickettsiae.

Nucleotide sequence of the Rickettsia prowazekii ATP/ADP translocase-encoding gene

The Rickettsia prowazekii ATP/ADP translocase (Tlc) gene (tlc), previously cloned in Escherichia coli was localized to a 1.6-kb chromosomal fragment. Nucleotide sequence analysis of this fragment revealed an open reading frame of 1494 bp that could encode a hydrophobic protein of 497 amino acids (aa) with an Mr of 56,668. Analysis of the deduced aa sequence revealed that it contained twelve potential membrane-spanning regions. Comparisons between the deduced aa sequence of the R. prowazekii ATP/ADP Tlc and the sequences of mitochondrial (mt) Tlc revealed no detectable homologies between the rickettsial and mt sequences. The major protein synthesized in E. coli minicells containing the rickettsial gene exhibited and Mr of approx. 34,000.

Conserved Response Regulator CtrA and IHF Binding Sites in the α-Proteobacteria Caulobacter crescentus and Rickettsia prowazekii Chromosomal Replication Origins

CzcR is the Rickettsia prowazekii homolog of the Caulobacter crescentus global response regulator CtrA. CzcR expression partially compensates for developmental defects in ctrA mutant C. crescentus cells, and CzcR binds to all five CtrA binding sites in the C. crescentus replication origin. Conversely, CtrA binds to five similar sites in the putative R. prowazekii replication origin (oriRp). Also, Escherichia coli IHF protein binds over a central CtrA binding site in oriRp. Therefore, CtrA and IHF regulatory proteins have similar binding patterns in both replication origins, and we propose that CzcR is a global cell cycle regulator in R. prowazekii.

DNA microarray analysis of the heat shock transcriptome of the obligate intracytoplasmic pathogen Rickettsia prowazekii.

ABSTRACT Here we present the first oligonucleotide DNA microarray analysis of global gene expression changes in the obligate intracytoplasmic pathogen Rickettsia prowazekii using temperature upshift as a model stress condition, and we describe a methodology for isolating highly purified rickettsial RNA. In toto, 23 transcripts were significantly increased by temperature upshift (≥2.0-fold; P < 0.05), and no transcripts demonstrated reproducible decreases. Array results for three heat shock-inducible mRNAs were confirmed using quantitative reverse transcription-PCR.

Rickettsial metK-Encoded Methionine Adenosyltransferase Expression in an Escherichia coli metK Deletion Strain

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.