The 1,995,275-bp genome of Coxiella burnetii, Nine Mile phase I RSA493, a highly virulent zoonotic pathogen and category B bioterrorism agent, was sequenced by the random shotgun method. This bacterium is an obligate intracellular acidophile that is highly adapted for life within the eukaryotic phagolysosome. Genome analysis revealed many genes with potential roles in adhesion, invasion, intracellular trafficking, host-cell modulation, and detoxification. A previously uncharacterized 13-member family of ankyrin repeat-containing proteins is implicated in the pathogenesis of this organism. Although the lifestyle and parasitic strategies of C. burnetii resemble that of Rickettsiae and Chlamydiae, their genome architectures differ considerably in terms of presence of mobile elements, extent of genome reduction, metabolic capabilities, and transporter profiles. The presence of 83 pseudogenes displays an ongoing process of gene degradation. Unlike other obligate intracellular bacteria, 32 insertion sequences are found dispersed in the chromosome, indicating some plasticity in the C. burnetii genome. These analyses suggest that the obligate intracellular lifestyle of C. burnetii may be a relatively recent innovation.

After repeated passages through embyronated eggs, the Nine Mile strain of Coxiella burnetii exhibits antigenic variation, a loss of virulence characteristics, and transition to a truncated lipopolysaccharide (LPS) structure. In two independently derived strains, Nine Mile phase II and RSA 514, these phenotypic changes were accompanied by a large chromosomal deletion (M. H. Vodkin and J. C. Williams, J. Gen. Microbiol. 132:2587-2594, 1986). In the work reported here, additional screening of a cosmid bank prepared from the wild-type strain was used to map the deletion termini of both mutant strains and to accumulate all the segments of DNA that comprise the two deletions. The corresponding DNAs were then sequenced and annotated. The Nine Mile phase II deletion was completely nested within the deletion of the RSA 514 strain. Basic alignment and homology studies indicated that a large group of LPS biosynthetic genes, arranged in an apparent O-antigen cluster, was deleted in both variants. Database homologies identified, in particular, mannose pathway genes and genes encoding sugar methylases and nucleotide sugar epimerase-dehydratase proteins. Candidate genes for addition of sugar units to the core oligosaccharide for synthesis of the rare sugar 6-deoxy-3-C-methylgulose (virenose) were identified in the deleted region. Repeats, redundancies, paralogous genes, and two regions with reduced G؉C contents were found within the deletions.Coxiella burnetii is an obligately parasitic bacterium that replicates within phagolysosomes of eukaryotic hosts (1,16,27). This organism is the causative agent of Q fever in humans. It is endemic in many species of domestic animals, especially sheep, goats, and cattle. C. burnetii is extremely infectious; inhalation of one organism is enough to initiate an acute illness in a guinea pig (32). In humans, the disease usually is a moderate to severe flu-like illness with headache, fever and chills, malaise, myalgia, and anorexia or an atypical pneumonia with a dry cough (26). Recrudescence and chronic illness, usually in the form of hepatitis or intractable endocarditis, are the most serious manifestations of the disease (24,25,28,55). Humans usually contract Q fever by inhaling contaminated dust, often in barnyard, stockyard, or abattoir settings (34, 45).Some strains of C. burnetii have been observed to undergo variation in surface antigens (18,40). Continual passage of the Nine Mile strain (the tick-derived United States prototype strain) through immunocompetent hosts (306 successive passages in guinea pigs) apparently maintained the organism in its original, native (wild-type) antigenic form. However, eight subsequent passages through embryonated eggs resulted in the emergence of organisms that were easily distinguished serologically from the original isolate (40). The new phenotype reacted strongly with complement-fixing antibody found in acute-phase serum, compared with the lack of a reaction demonstrated by the guinea pig-passaged antigenic form (40). The two antigenic types differed ...

Direct analysis in real time (DART) is implemented on a time-of-flight (TOF) mass spectrometer, and used for the generation of fatty acid methyl esters (FAMEs) ions from whole bacterial cells.

. In the present study, an ars replicon was used to transform C. burnetii to ampicillin resistance. Plasmid pSKO(؉)1000 contained the C. burnetii ars sequence cloned into a ColE1-type replicon encoding ␤-lactamase. pSKO(؉)1000 was introduced into C. burnetii by electroporation. Ampicillin-resistant cells were selected, and survivors were examined for the transformed genotype by Southern hybridization. Transformants stably maintained the pSKO(؉)1000 bla DNA sequence in the chromosome as a result of homologous recombination. The recombination event resulted in the duplication of the 5.8-kb ars sequence in the C. burnetii chromosome. The bla gene was also located in an episome. However, an ampicillin resistance plasmid lacking the C. burnetii ars sequence did not stably transform C. burnetii. A biological assay analyzing ␤-lactamase activity of C. burnetii transformants during acid activation in vitro provided evidence for expression of the bla (␤-lactamase) gene.Coxiella burnetii is the etiological agent of human Q fever (9). It is an obligate intracellular bacterium replicating within the acidic phagolysosomes of eukaryotic cells (1,4,15,24). Although C. burnetii becomes metabolically active and synthesizes protein and DNA during in vitro acid activation assays, axenic growth has not been observed (7,15,43). This obligate intracellular parasitism poses difficulties in studying virulence factors associated with this organism. Classical genetic studies involve the mutation of genes to eliminate a virulent phenotype and then complementation using DNA from wild-type organisms to restore virulence; this approach ascertains the contribution of specific genes to virulence. However, these types of studies have been unavailable for C. burnetii, since genetic transformation of this organism has not yet been demonstrated.Information on enzymes produced by C. burnetii and on gene structure within the chromosome and the endogenous plasmid QpHI has been obtained by the cloning and expression of C. burnetii genes in Escherichia coli (19). The use of E. coli as a host for C. burnetii genes was also implemented for the cloning of the C. burnetii chromosomal origin of DNA replication. These origin search techniques resulted in the isolation of a 5.8-kb C. burnetii chromosomal DNA fragment which initiates plasmid DNA replication within E. coli (36). The minimal ars sequence required for plasmid replication in an E. coli polA strain is 403 bp (6, 36); it demonstrates limited similarity to origins of other bacteria (Fig. 1). The ars sequence contains two consensus sequences for the binding of DnaA, an AϩT-rich region, and a potential binding site for integration host factor and for factor of inversion stimulation; these sequences are characteristic of bacterial chromosomal origins (3,12,28). The open reading frames flanking one side of the C. burnetii ars sequence demonstrate similarity to genes located in bacterial origin regions; these open reading frames include rnpA and rpmH genes and genes encoding 9K and 60K proteins (Fig. 1). ...

We describe a fatal pediatric case of Rocky Mountain spotted fever in Panama, the first, to our knowledge, since the 1950s. Diagnosis was established by immunohistochemistry, PCR, and isolation of Rickettsia rickettsii from postmortem tissues. Molecular typing demonstrated strong relatedness of the isolate to strains of R. rickettsii from Central and South America.

Although Q fever is considered enzootic in the United States, surveillance for human Q fever has been historically limited. From 1978 through 1999, 436 cases (average = 20 per year) of human Q fever were reported. After Q fever became nationally reportable in 1999, 255 human Q fever cases (average = 51 per year) were reported with illness onset during 2000 through 2004. The median age of cases was 51 years, and most cases were male (77%). The average annual incidence of Q fever was 0.28 cases per million persons, and was highest in persons 50-59 years of age (0.39 cases per million). State-specific incidence ranged from a high of 2.40 cases per million persons in Wyoming, to 0 cases in some states. Since Q fever became reportable, case reports have increased by more than 250%. Surveillance for Q fever is essential to establish the distribution and magnitude of disease and to complement U.S. bioterrorism preparedness activities.

Background: Coxiella burnetii contains the IS1111 transposase which is present 20 times in the Nine Mile phase I (9Mi/I) genome. A single PCR primer that binds to each IS element, and primers specific to a region ~500-bp upstream of each of the 20 IS1111 elements were designed. The amplified products were characterized and used to develop a repetitive element PCR genotyping method.

A population of free, native ribosomal 40S subunits, that do not react with 60S subunits to form 80S ribosomes, has been identified in the postmicrosomal fraction of rat liver homogenates. A protein (IF-3) has been purified from high salt (0.88 M KCI) extracts of native 40S subunits by gradient centrifugation and by ammonium sulfate fractionation; it prevents the reassociation of subunits and to a limited extent dissociates ribosomes to subunits. The activity is measured by ultracentrifugation of the reaction products on linear sucrose gradients, or with an assay developed in this laboratory that couples dissociation with the 60S-specific peptidyltransferase reaction; the latter procedure measures the amount of 60S subunits released from ribosomes or remaining in incubations in the presence of IF-3. Dissociation factor activity is recovered from most of the particles that are resolved by zonal centrifugation of the total "native subunits" obtained from the postmicrosomal fraction; the highest concentration of IF-3, however, appears to be associated with native 40S subunits. The purified dissociation factor IF-3 is composed of about ten polypeptides and the molecular weight is estimated to be between 500 000 and 700 000, on the basis of glycerol and cesium chloride gradient centrifugation. When purified 40S subunits react with IF-3 or when 80S ribosomes are dissociated by IF-3, a product is formed which is dependent on the concentration of the protein factor and has the characteristics of a 40SIF-3 complex; centrifugation of the complex on sucrose and cesium chloride gradients suggests that the complex consists of 1 equiv of each of the two components. Although dissociation factor IF-3 appears to react in a specific manner with free or ribosome-associated 40S subunits, the reaction with subunits differs in several respects from that with ribosomes. The dissociation factor also appears to interact with 60S subunits but multiple complexes are formed, some with more than 1 IF-3 equiv per 60S particle. The IF-3 converts 40S dimers (55S particles) to the 40S-IF-3 complex and dissociates free, native 80S particles present in the postmicrosomal fraction, but it does not affect polysome-associated ribosomes engaged in protein synthesis.