David O. Wood

The citrate synthase-encoding gene of Rickettsia prowazekii is controlled by two promoters.

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.

Genetic systems for studying obligate intracellular pathogens: an update.

Rapid advancements in the genetic manipulation of obligate intracellular bacterial pathogens have been made over the past two years. In this paper we attempt to summarize the work published since 2011 that documents these exciting accomplishments. Although each genus comprising this diverse group of pathogens poses unique problems, requiring modifications of established techniques and the introduction of new tools, all appear amenable to genetic analysis. Significantly, the field is moving forward from a focus on the identification and development of genetic techniques to their application in addressing crucial questions related to mechanisms of bacterial pathogenicity and the requirements of obligate intracellular growth.

Dissecting the Rickettsia prowazekii Genome: Genetic and Proteomic Approaches

Abstract: The obligate nature of Rickettsia prowazekii intracellular growth places severe restrictions on the analysis of rickettsial gene function and gene expression. Fortunately, this situation is improving as methods for the genetic manipulation and proteomic analysis of this fascinating human pathogen become available. In this paper, we review the current status of rickettsial genetics and the isolation of rickettsial mutants using a genetic approach. In addition, the examination of rickettsial gene expression through characterization of the rickettsial proteome will be described. This will include a description of a high‐throughput, accurate mass approach that has identified 596 rickettsial proteins in a complex rickettsial protein sample.

Isolation and characterization of the Rickettsia prowazekii gene encoding the flavoprotein subunit of succinate dehydrogenase.

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.

Isolation and characterization of the gene coding for the major sigma factor of Rickettsia prowazekii DNA-dependent RNA polymerase

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.

Sequence analysis of the Rickettsia prowazekii gyrA gene.

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.

Differential Proteomic Analysis of Rickettsia prowazekii Propagated in Diverse Host Backgrounds

ABSTRACT The obligate intracellular growth of Rickettsia prowazekii places severe restrictions on the analysis of rickettsial gene expression. With a small genome, predicted to code for 835 proteins, identifying which proteins are differentially expressed in rickettsiae that are isolated from different hosts or that vary in virulence is critical to an understanding of rickettsial pathogenicity. We employed a liquid chromatography (LC)-linear trap quadrupole (LTQ)-Orbitrap mass spectrometer for simultaneous acquisition of quantitative mass spectrometry (MS)-only data and tandem mass spectrometry (MS-MS) sequence data. With the use of a combination of commercially available algorithms and in-house software, quantitative MS-only data and comprehensive peptide coverage generated from MS-MS were integrated, resulting in the assignment of peptide identities with intensity values, allowing for the differential comparison of complex protein samples. With the use of these protocols, it was possible to directly compare protein abundance and analyze changes in the total proteome profile of R. prowazekii grown in different host backgrounds. Total protein extracted from rickettsiae grown in murine, tick, and insect cell lines or hen egg yolk sacs was analyzed. Here, we report the fold changes, including an upregulation of shock-related proteins, in rickettsiae cultivated in tissue culture compared to the level for rickettsiae harvested from hen yolk sacs. The ability to directly compare, in a complex sample, differential rickettsial protein expression provides a snapshot of host-specific proteomic profiles that will help to identify proteins important in intracellular growth and virulence.

Characterization of the gene coding for the Rickettsia prowazekii DNA primase analogue

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.

Rickettsia prowazekii and ATP/ADP Translocase

Rickettsia prowazekii, the etiologic agent of epidemic typhus, grows and multiplies exclusively within the cytoplasm of a eucaryotic host cell. One of the advantages provided by this unique environmental niche is the abundance of nutrients, including many preformed metabolites, which are readily available to the growing rickettsiae. R. prowazekii has been shown to transport and metabolize a number of these preformed metabolites, including NAD, UDPG, AMP, ADP and ATP.L-4 The ability to transport, rather than synthesize, key metabolites is one example of how R. prowazekii takes advantage of the unique environment eucaryotic cytoplasm offers. The rickettsial ATP/ADP t r a n ~ l o c a s e ~ . ~ is an obligate exchange transport system similar in function to the mitochondrial ADP/ATP translocases in that for every ADP or ATP molecule transported into the rickettsiae, an ATP or ADP must also be expelled. The rickettsial translocase is specific for ADP and ATP, has no energy requirement, and is unaffected by the classic inhibitors of the mitochondria1 translocases, atractyloside and bongkrekic acid. Inside the host cell, the rickettsiae presumably transport in ATP in exchange for rickettsial ADP. The net result of this exchange is the transport of a high-energy phosphate bond into the rickettsial cell. The mechanism which maintains this directionality is not fully understood; however, the transport of ATP over ADP is favored when the phosphate concentration of the surrounding medium is low.6 The gene encoding the rickettsial ATP/ADP translocase has been cloned and expressed in Escherichia coli by Krause et aL7 The cloned rickettsial translocator exhibited all the same characteristics as previously described in R. prowazekii, including specificity for ADP and ATP, no energy requirement, and insensitivity to atractyloside and bongkrekic acid. Although the translocase activity was easily measured in E. coli, attempts to identify the translocase protein product were unsuccessful. The gene coding for the ATP/ADP translocase (tlc) was subcloned

Screening the Rickettsia prowazekii genome for rpoD homologs.

The procaryotic DNA-dependent RNA polymerase holoenzyme consists of a, p , p‘, and IT subunits. The core enzyme (az,p,p’) is responsible for the elongation phase of transcription, while the sigma factor (u) is an essential element in the initiation process. In addition to a principal sigma factor, many bacteria possess one or more minor sigma factors. The minor sigma factors are classically involved in stress-related phenomenon such as “heat shock” and spore formation.’ The various sigma factors differ in promoter recognition and thereby either initiate the transcription of additional genes or alter the initiation frequency of constitutively expressed genes. The holoenzyme has a markedly decreased ability to bind to random sequences of DNA and a drastically increased ability to bind to specific sequences of DNA compared to that of the core enzyme alone. These specific DNA sequences are identified by the classical characteristics of a promoter region. Once the holoenzyme binds to the DNA template and RNA synthesis has been initiated, the sigma factor is quickly released from the complex and can then be recycled. The principal sigma factor of Escherichia coli is the product of the rpoD gene and has a molecular weight of 70,000. The protein contains at least two functional domains, one for binding to the core enzyme and another for binding to the DNA template. The core-binding domain can be subdivided into two subdomains, A and B. Unlike the A subdomain, the B subdomain is highly conserved in the principal sigma factors of E. coli, Salmonella typhimuriurn, and Bacillus subtilis but not in the minor sigma factors.* The 33 amino acids associated with the B subdomain are 100% and 91% identical between E. coli and S . typhimurium and between E. coli and B. subtilis, respectively. In order to identify rpoD gene homologs in other species, a 29-base synthetic oligonucleotide probe was constructed by Tanaka et a1.2 The sequence of the probe was derived from the highly conserved subdomain B, and the problem of codon degeneracy was addressed by utilizing inosine: