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Recent studies have demonstrated that Rickettsia prowazekii can regulate transcription of selected genes at the level of initiation. However, little information concerning the existence of operons and coordinate gene regulation in this obligate intracellular parasitic bacterium is available. To address these issues, we have focused on the rpoD gene linkage group (greA-open reading frame 23 [ORF23]-dnaG-rpoD), which includes the rickettsial analog (ORF23-dnaG-rpoD) of the major macromolecular synthesis operon (MMSO). The rickettsial MMSO consists of an ORF coding for a protein of unknown function the structural genes for DNA primase (dnaG) and the major sigma factor of RNA polymerase (rpoD). RNase protection assays (RPA) were used to determine if these genes are organized into an operon controlled by multiple promoters and the quantities of transcripts produced by these genes relative to each other. RPA with a probe spanning the 270-base greA-ORF23 intervening region identified a putative transcriptional promoter within the intervening sequence. Multiple RPA probes spanning the next 4,041 bases of the linkage group demonstrated the presence of a continuous transcript and thus the existence of an operon. A probe spanning the dnaG-rpoD region revealed that two additional mRNA fragments were also protected, which enabled us to identify additional putative promoters for rpoD within dnaG. Primer extension determined that the 5 ends of the three transcripts consist separately of adenine (located 227 bases upstream of ORF23) and uracil and adenine (located 336 and 250 bases upstream of rpoD, respectively). Quantitation of transcripts produced by the three ORFs determined the relative amounts of transcripts (ORF23 to dnaG to rpoD) to be 1:2.7:5.1.The obligate intracellular pathogen Rickettsia prowazekii is a bacterium with gram-negative morphology and the etiologic agent of epidemic typhus. The ability of this organism to invade a host cell and grow freely in the cytoplasm until rickettsia-induced cell lysis occurs is the basis for its pathogenicity. Despite R. prowazekii's nutrient-rich environment and numerous pathways to exploit this environment, the organism maintains an 8-to 12-h replication time (35). This relatively slow replication time can be attributed to any number of factors, with macromolecular synthesis (MMS) mechanisms being possible targets of regulation. These mechanisms, of course, include DNA replication, transcription of the various RNA species, and protein synthesis processes. Within the plethora of regulatory strategies controlling these fundamental processes in bacteria, regulation of gene expression by altering the number of translatable transcripts is by far the most common strategy.Although comparatively little is known about the in vivo regulatory mechanisms employed by R. prowazekii, our understanding of this pathogen's metabolic and molecular structures and capabilities is increasing. R. prowazekii has a genome size of 1.1 Mb (13), and an initial survey of gene content and coding capacity has...