Comparative ultrastructural study on the cell envelopes of Rickettsia prowazekii, Rickettsia rickettsii, and Rickettsia tsutsugamushi.
Rickettsia tsutsugamushi differs from other rickettsiae in its cell envelope organization. The differences were made evident through a comparative study of the outer envelope of R. tsutsugamushi, R. prowazekii, and R. rickettsii by electron microscopy. (+info
Surface proteins of typhus and spotted fever group rickettsiae.
Six proteins, previously established as major constituents of intact organisms, were identified in cell envelopes obtained from intrinsically radiolabeled Rickettsia prowazekii. Extrinsic radioiodination of intact organisms conducted at 0.5 micronM iodide indicated that protein 4 was the most peripheral, although protein 1 also had reactive groups exposed on the surface of the organisms. A 10-fold increase in iodide concentration resulted in labeling of protein 2, and at 50 micronM iodide, all six major proteins were radiolabeled. Similar selective labeling was not achieved with R. conorii. Analysis of both typhus and spotted fever group organisms radiolabeled with galactose suggested that carbohydrate was associated with proteins 1, 3, and 4. Typhus soluble antigen included all major proteins except protein 2, which remained attached to particulate rickettsiae after ether extraction. Protein 4 appeared to be prominent in the surface topography of R. prowazekii, was a component of soluble antigen and may have an important role in rickettsiae-host interactions. (+info
Phospholipid composition of Rickettsia prowazeki grown in chicken embryo yolk sacs.
The phospholipid composition and phospholipid fatty acid composition of purified Rickettsia prowazeki were determined. The lipid phosphorous content was 6.8 +/- 1.3 microgram/mg of total rickettsial protein. The major phospholipid was phosphatidylethanolamine (60 to 70%); phosphatidylglycerol constituted 20%, and phosphatidylcholine constituted 15%. Small amounts of phosphatidylserine and cardiolipin were detected. The principal fatty acids were 18:1, 16:1, and 16:0. The fatty acid composition of the phosphatidylcholine in the rickettsial extracts was very different than that of the other rickettsial phosphatides and very similar to that of normal yolk sac phosphatidylcholine. The specific of the phosphatidylcholine of rickettsiae grown in the presence of 32P was markedly lower than that of phosphatidylethanolamine and phosphatidylglycerol. It is suggested that the phosphatidylcholine in the rickettsial extract is yolk sac derived and either tightly absorbed or exchanged into the rickettsial membrane. (+info
Absence of hydrogen peroxide production by or catalase action in Rickettsia prowazeki.
Glutamic acid oxidation by Rickettsia prowazeki is not accompanied by hydrogen peroxide generation, nor is added hydrogen peroxide degraded, as measured by a manometric technique. (+info
Rickettsia prowazekii transports UMP and GMP, but not CMP, as building blocks for RNA synthesis.
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. (+info
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. (+info
Nucleotide sequence of the gene and features of the major outer membrane protein of a virulent Rickettsia prowazekii strain.
We have determined the nucleotide sequence of the gene for a major outer membrane protein (MOMP) of apparent molecular weight 29.5 kD of the virulent Breinl strain of Rickettsia prowazekii. The gene contains an open reading frame (ORF) that encodes a 282-amino-acid polypeptide with a calculated molecular mass of 31549 daltons. A signal-like peptide sequence is found at the deduced N terminus. A heterologous 29.5-kD antigen expressed in Escherichia coli was shown to be secreted into the periplasm. A database search for similar protein sequences revealed considerable homology of the polypeptide with the E. coli peptidyl-prolyl cis/trans isomerase and related proteins of the parvulin family. The genes for MOMP of the virulent Breinl and EVir strains and the vaccine Madrid E strain were amplified using specific primers and cloned into expression vector pQE-30. We found that the polypeptides encoded by the recombinant DNAs do not differ in SDS-PAGE mobility, while the native MOMP of the Breinl strain is known to be different from the corresponding proteins of the Madrid E and EVir strains. Furthermore, no differences within the ORF for the 29.5-kD proteins of the three strains were found by restriction endonuclease analysis of polymerase chain reaction (PCR) products. A possible role of parvulin-like protein (Plp) in the virulence of epidemic typhus agent and the nature of interstrain differences are discussed. Near the plp gene on the opposite strand, an origin of the gene that codes for the SecA subunit of a preprotein translocase was found. (+info
Gene synthesis, bacterial expression and purification of the Rickettsia prowazekii ATP/ADP translocase.
The Rickettsia prowazekii ATP/ADP translocase (Tlc) is the first member of a new family of ATP/ADP exchangers that includes both prokaryotic and eukaryotic proteins. We optimized the codon usage for expression of tlc in Escherichia coli by means of gene synthesis, expressed the synthetic gene in E. coli, and purified a modified Tlc that contained a C-terminal tag of 10 consecutive histidine residues by immobilized metal affinity chromatography. Although codon usage in R. prowazekii is very different from E. coli, the optimization of the codon usage by itself was insufficient to improve expression. However, the change of the cloning vector from pET11a to pT7-5 led to a 3-10-fold increase in the specific ATP transport rate by cells expressing the synthetic construct. The authenticity of the purified protein was confirmed by N-terminal amino acid sequencing and a matrix assisted laser desorption/ionization mass spectrometry. (+info