Members of the genus Rickettsia, including Rickettsia prowazekii, the etiological agent of epidemic typhus, are unique, even amongst the obligate intracellular bacterial parasites. Rickettsiae grow directly in the cytoplasm, rather than within a vacuole in the cytoplasm of their eukaryotic host cells. The investigators are committed to understanding the mechanisms by which R. prowazekii copes with the problems and exploits the opportunities of this unique niche. This proposal is focused on two fundamental problems of rickettsiology: i) how ATP/ADP cross the rickettsial membrane on a translocase that is present only in obligate intracellular bacteria and, despite its lack of sequence homology to it, functions like the ADP/ATP translocase in the mitochondrion; and ii) how the rickettsiae, which have a milieu exterieur that is our milieu interieur, regulate their transport and metabolic activity at the transcriptional level. I. Transcriptional regulation in R. prowazekii. Although rickettsiae can regulate the activity of their enzymes, whether there is transcriptional control of the amount of enzyme in R. prowazekii is unknown. To determine if R. prowazekii has transcriptional regulation of the tlc (ATP/ADP translocase), gltA (citrate synthase), and rrs(16S rRNA) genes and to define the properties of this control mechanism are the goals of this specific aim. The characterization will include: 1) Sequence analysis of the upstream (regulatory) regions of the tlc, gltA, and rrs genes. 2) Determination of the transcription initiation sites and promoters of the tlc, gltA, and rrs genes. 3) Examination of control of 16S rRNA production. 4) Investigation of the level of transcriptional activity on the gltA and tlc genes at different growth conditions. 5) Identification of the transcription regulatory DNA elements, the signal molecules (cofactors), and the transcription regulator proteins. II. Characterization of the ATP/ADP translocase of R. prowazekii.
This specific aim has four parts. 1) The investigators will overexpress the rickettsial ATP/ADP translocase (tlc) in E. coli. 2) They will purify the tlc from E. coli strains overexpressing this rickettsial transport protein so that it can be N-terminal sequenced, peptide mapped, and reconstituted into active proteoliposomes. 3) They will determine the topology of the tlc, that is, the manner in which this amphipathic protein is laced back and forth through the membrane. The topology studies will employ four methodologies: phoA fusions, specific antipeptide antibody accessibility, vectorial labeling, and protease accessibility. 4) They will determine the active sites of this obligate exchange transport system, that is, the sites which bind the transported substrates ATP/ADP. These active site studies will employ both covalently bonded affinity reagents (pyridoxal-5-phosphate and azidoATP) and site specific mutagenesis based on the conservation of sequence between R. prowazekii, R. rickettsii and chlamydia.
Showing the most recent 10 out of 52 publications
