Small subunit rRNA sequences have been determined for representative strains of six species of the family Rickettsiaceae: Rickettsia rickettsii, Rickettsia prowazekii, Rickettsia typhi, Coxiella burnetii, Ehrlichia risticii, and Wolbachia persica. The relationships among these sequences and those of other eubacteria show that all members of the family Rickettsiaceae belong to the so-called purple bacterial phylum. The three representatives of the genus Rickettsia form a tight monophyletic cluster within the a subdivision of the purple bacteria. E. risticii also belongs to the a subdivision and shows a distant yet specific relationship to the genus Rickettsia. However, the family as a whole is not monophyletic, in that C. burnetii and W. persica are members of the -y subdivision. The former appears to show a specific, but rather distant, relationship to the genus Legionella.The order Rickettsiales (32) comprises a collection of procaryotes that share the property of intimate association with eucaryotic cells. The relationship in most cases is obligate intracellular parasitism, although a few of these bacteria have been grown in complex host-cell-free culture media. Although this order contains notorious pathogens of humans and animals, some of these bacteria cause no obvious damage to their host, and the relationship can be regarded as commensal. The Rickettsiales are clearly separate from the Chlamydiales (17), a narrowly defined group of energy-parasitizing, obligately intracellular bacteria. Certain fastidious parasites of plant vascular tissues and arthropods, sometimes referred to as rickettsialike (5), are not included in either category. Also, the highly heterogeneous group of bacteria that have established an endosymbiotic relationship with their hosts and for the most part have not been cultivated, including the hydrothermal-vent-associated symbionts (3,4,21), are viewed as distinct from the Rickettsiales (32) in the latest edition of Bergey's Manlual of Systematic Bacteriology.The order Rickettsiales contains three families: Rickettsiaceae, Bartonellaceae, and Anaplasmataceae. Our present study is confined to the family Rickettsiaceae, which is divided into three tribes: Rickettsieae, Ehrlichieae, and Wolbachieae (32). In the family Rickettsiaceae, rigorous criteria of classical taxonomy have been applied to the definition of most species and, in some but not in all cases, the genera. For example, good evidence was obtained by phenotypic analysis, DNA base ratio determinations, and DNA-DNA hybridization studies that in the genus Rickettsia, members of the typhus and spotted fever groups are related (32). However, the degree of relatedness of these microorganisms to the scrub typhus rickettsia, Rickettsia tsutsugamushi, remains unknown. Similarly, phenotypic analysis links the monocytic erhlichiae (Ehrlichia canis, Ehrlichia sennetsu, and Ehrlichia risticii) to each other (10,22,23), but their relationships to the granulocytic ehrlicheae * Corresponding author. t Present address: Gene-Trak Systems,

Thirty-two isolates of Coxiella burnetii collected from various hosts ranging from arthropods to man were compared by restriction endonuclease (RE) digestion patterns of chromosomal DNA using SDS-PAGE. SDS-PAGE provided better DNA fragment separation than agarose gel electrophoresis and enabled the differentiation of these isolates into six distinct groups on the basis of DNA restriction fingerprints. Two groups of chronic disease isolates could be distinguished, each having unique RE digestion patterns of chromosomal DNA. Three similar but distinct RE digestion patterns were seen among the group of acute disease isolates. Three additional isolates included in this study exhibited a unique RE digestion pattern and also had a unique plasmid type, designated QpDG. DNA-DNA hybridization on selected isolates quantified the relatedness between several groups and supported the classification of these groups as distinct strains.

Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular bacterium proliferating within the harsh environment of the phagolysosome. Mechanisms controlling trafficking to, and survival of pathogens within, the phagolysosome are unknown. Two distinct morphological variants have been implicated as playing a role in C. burnetii survival. The dormant small-cell variant (SCV) is resistant to extracellular stresses and the more metabolically active large-cell variant (LCV) is sensitive to environmental stresses. To document changes in the ratio of SCVs to LCVs in response to environment, a protein specific to SCV, ScvA, was quantitated. During the first 2 h after internalization of C. burnetii by J774A.1 cells, the level of ScvA decreased, indicating a change from a population containing primarily SCVs to one containing primarily LCVs. In vitro experiments showed that 2 h of incubation at pH 5.5 caused a significant decrease in ScvA in contrast to incubation at pH 4. Measuring in vitro internalization of [35 S]methionine-[ 35 S]cysteine in response to pH, we found the uptake to be optimal at pH 5.5. To explore the possibility that after uptake C. burnetii was able to delay phagolysosomal fusion, we used thorium dioxide and acid phosphatase to label phagolysosomes during infection of J774A.1 cells. We determined that viable C. burnetii was able to delay phagolysosomal fusion. This is the first time that a delay in phagolysosomal fusion has been shown to be a part of the infection process of this pathogenic microorganism.

The obligate intracellular bacterium Coxiella burnetii is the etiological agent of acute Q-fever and chronic endocarditis in humans and of several zoonotic infections. The DNA from a variety of these disease isolates was compared for homology to the plasmid QpHl, found in the Nine Mile strain. Three patterns of homology were found in these isolates, i.e., one pattern identical to that of QpHl, one common to several endocarditis isolates and goat abortion isolates, and one common to the remaining group of endocarditis isolates. Plasmid DNA from the endocarditis-abortion isolate group, designated QpRS, was mapped by restriction enzyme analysis and compared with QpHl. These data show that QpRS was 2 to 3 kilobase pairs larger, contained DNA not found in QpHl, but was not generated from QpHl by a single insertional event. Isolation of plasmid DNA from the second endocarditis group of isolates was not successful and may indicate that the plasmid has integrated into the chromosome. This analysis provides the first clear evidence that differences exist between C. burnetii isolates which cause various diseases, indicating that different C. burnetii strains may have unique virulence characteristics. Coxiella burnetii is an obligate intracellular parasite that causes Q-fever (2). The disease normally presents as an acute febrile illness, with recovery occurring in 1 to 4 weeks. C. burnetii has also been implicated as the etiological agent in a number of cases of chronic endocarditis in humans. These infections have been considered to be due to the compromised nature of the host during infection and not to a specific property of the pathogen (6, 7, 11). C. burnetii is also a zoonotic pathogen, most notably responsible for late-term abortions in several domestic animals (e.g., goat and sheep; 1, 9). Comparative analyses of organisms isolated from hosts for each of these diseases have not indicated that there are any specific C. burnetii variants able to cause a particular

Coxiella burnetii undergoes a poorly defined developmental cycle within phagolysosomes of eukaryotic host cells. Two distinct developmental forms are part of this cycle: a small-cell variant (SCV) and large-cell variant (LCV). Ultrastructurally, the SCV is distinguished from the LCV by its smaller size and condensed chromatin. At a molecular level, little is known about morphogenesis in C. burnetii, and no proteins specific to the SCV have been identified. Preparative isoelectric focusing was conducted to purify basic proteins possibly involved in SCV chromatin structure. A predominant protein of low M(r) was present in the most basic fraction, eluting with a pH of approx. 11. Degenerate deoxyoligonucleotides corresponding to the N-terminal sequence of this protein were used to recover a cosmid clone from a C. burnetii genomic library. Nucleotide sequencing of insert DNA revealed an open reading frame designated scvA (Small-Cell-variant protein A) with coding potential for a 30 amino acid protein (ScvA) with a predicted M(r) of 3610. ScvA is 46% arginine plus 46% glutamine with a predicted pl of 12.6. SDS-PAGE and silver staining of lysates of SCV and LCV purified by caesium chloride-equilibrium density centrifugation revealed a number of proteins unique to each cell type. Immunoblot analysis with ScvA antiserum demonstrated the presence of ScvA only in the SCV. By Immunoelectron microscopy, ScvA antiserum labelled only the SCV, with the label concentrated on the condensed nucleoid. In addition, ScvA bound double-stranded DNA in gel mobility-shift assays. A 66% reduction in the mean number of gold particles per Coxiella call was observed at 12 h post-infection when compared with the starting inoculum. Collectively, these data suggest that synthesis of ScvA is developmentally regulated, and that the protein may serve a structural or functional role as an integral component of the SCV chromatin. Moreover, degradation of this protein may be a necessary prerequisite for morphogenesis from SCV to LCV.