Members of the genusRickettsia, including Rickettsia prowazekii, the etiological agent ofepidemic typhus, are unique, even amongst the obligate intracellularbacterial parasites. Rickettsiae grow directly in the cytoplasm, ratherthan within a vacuole in the cytoplasm of their eukaryotic host cells. The investigators are committed to understanding the mechanisms by whichR prowazekii copes with the problems and exploits the opportunities ofthis unique niche. This proposal is focused on two fundamental problemsof rickettsiology: i how ATPADP cross the rickettsial membrane on atranslocase that is present only in obligate intracellular bacteria and, despite its lack of sequence homology to it, functions like the ADPATPtranslocase in the mitochondrion; and ii how the rickettsiae, whichhave a milieu exterieur that is our milieu interieur, regulate theirtransport and metabolic activity at the transcriptional level. I Transcriptional regulation in R prowazekii. Although rickettsiae canregulate the activity of their enzymes, whether there is transcriptionalcontrol of the amount of enzyme in R prowazekii is unknown. To determineif R prowazekii has transcriptional regulation of the tlc ATPADPtranslocase, gltA citrate synthase, and rrs16S rRNA genes and todefine the properties of this control mechanism are the goals of thisspecific aim. The characterization will include: 1 Sequence analysisof the upstream regulatory regions of the tlc, gltA, and rrs genes. 2 Determination of the transcription initiation sites and promoters ofthe tlc, gltA, and rrs genes. 3 Examination of control of 16S rRNAproduction. 4 Investigation of the level of transcriptional activityon the gltA and tlc genes at different growth conditions. 5Identification of the transcription regulatory DNA elements, the signalmolecules cofactors, and the transcription regulator proteins. II Characterization of the ATPADP translocase of R prowazekii. Thisspecific aim has four parts. 1 The investigators will overexpress therickettsial ATPADP translocase tlc in Ecoli. 2 They will purify thetlc from E coli strains overexpressing this rickettsial transportprotein so that it can be N-terminal sequenced, peptide mapped, andreconstituted into active proteoliposomes. 3 They will determine thetopology of the tlc, that is, the manner in which this amphipathicprotein is laced back and forth through the membrane. The topologystudies will employ four methodologies: phoA fusions, specificantipeptide antibody accessibility, vectorial labeling, and proteaseaccessibility. 4 They will determine the active sites of this obligateexchange transport system, that is, the sites which bind the transportedsubstrates ATPADP. These active site studies will employ both covalentlybonded affinity reagents pyridoxal-5-phosphate and azidoATP and sitespecific mutagenesis based on the conservation of sequence between R prowazekii, R rickettsii and chlamydia.
My lab is studying mechanisms of infection and growth by the intracellular bacterium Rickettsia prowazekii. Rickettsiae, which are transmitted by arthropods, are the parasitic organisms that cause typhus and Rocky Mountain spotted fever. Rickettsiae cannot grow in any known artificial medium, Rickettsiae grow only within the cytoplasm of eukaryotic host cells. Over the past 25 years, we have been studying how rickettsiae enter the host cell, how they grow and divide in the cell, and how their metabolism has evolved to take advantage of this unusual environment -- cytoplasm. However, much of what Rickettsiae do in this unique ecological niche remains to be discovered. We have performed extensive studies on the biochemistry of the rickettsial membrane and its transport systems. Transport systems of obligate intracellular bacteria in general and the specific molecular properties of the transport system used to obtain ATP from the host cell cytoplasm are current projects. We are also constructing a DNA array so that when the rickettsia changes environments transcription of all 834 orfs (that's all!) can be evaluated at one time
Adenosine Diphosphate, Adenosine Triphosphate, Biological Signal Transduction, Escherichia Coli, Genetic Promoter Element, Genetic Transcription, Membrane Permeability, Nucleic Acid Sequence, Protein Transport, Ribosomal Rna, Rickettsia Prowazekii, Site Directed Mutagenesis, Transcription Factor, Transport Protein, Typhus
