Comparison of <i>Coxiella burnetii</i> Plasmids to Homologous Chromosomal Sequences Present in a Plasmidless Endocarditis‐Causing Isolatea

Savinelli, Elizabeth A.; Mallavia, Louis P.

doi:10.1111/j.1749-6632.1990.tb42262.x

Cited by 47 publications

(27 citation statements)

References 14 publications

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“…The detection of chromosomally integrated plasmid-homologous DNA fragments in C. burnetii isolate Scurry Q217 by hybridization experiments (8) suggests that this genetic information is of crucial importance for the organism. Unlike in other bacteria, where plasmid genes are believed to provide supplementary activity (7), plasmid sequences are supposed to have a critical function in C. burnetii, since they have been conserved in all isolates examined (7), even in the plasmidless isolate Scurry Q217.…”

Section: Discussionmentioning

confidence: 99%

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Plasmid-homologous sequences in the chromosome of plasmidless Coxiella burnetii Scurry Q217

et al. 1997

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Chromosomal DNA from Coxiella burnetii Scurry Q217 was screened for the presence of plasmid-homologous sequences. Total DNA from Scurry Q217 was digested with NotI, and the resulting DNA fragments were separated by contour-clamped homogeneous electric field pulsed-field gel electrophoresis (CHEF-PFGE). Following hybridization with biotin-labeled QpH1 plasmid as a probe, two DNA fragments of 40 and 170 kb were identified as targets. These fragments were cloned, and subclones containing QpH1-homologous sequences were completely sequenced. The physical mapping of DNA fragments was achieved by PCR with primers derived from adjacent fragments, and a total of 18,360 bp was sequenced. ORFs were different in size, 6 ORFs were newly generated, and 25 ORFs were lost. It was found that plasmid-homologous sequences in Scurry Q217 were of chromosomal origin.Coxiella burnetii, the only member of the genus Coxiella, is the causative agent of Q fever in humans as well as of abortions in domestic animals, especially ruminants. So far, different criteria have been applied for the classification of C. burnetii isolates. Samuel et al. (7) distinguish between so-called acute and chronic isolates that cause acute or chronic Q fever in humans, apparently depending on the plasmid type. However, recent experimental findings (3) and PCR analysis of C. burnetii strains isolated from patients exhibiting chronic Q fever (11) revealed that isolates containing either QpH1 or QpRS can cause endocarditis. Yu and Raoult (15) confirmed these results by serotyping isolates with monoclonal antibodies and postulated that the development of acute or chronic Q fever depends on the patient's condition and immune status rather than on the plasmid type of the causative agent. Nevertheless, plasmids are still considered to be of major importance concerning virulence factors, mechanisms of pathogenicity, and proliferation in the phagolysosomal compartment.Assuming that the plasmid-coded sequences are of essential importance for C. burnetii, the existence of the plasmidless C. burnetii isolate Scurry Q217 (8) was rather unexpected. However, it was shown that Scurry Q217 contains chromosomally integrated plasmid-homologous DNA sequences (8). In an attempt to understand the significance of plasmids in C. burnetii, we examined plasmidless isolates with chromosomally integrated plasmid-homologous DNA fragments. MATERIALS AND METHODSDNA-modifying enzymes. Restriction endonucleases and other DNA-modifying enzymes were purchased from Stratagene (Heidelberg, Germany) and Amersham (Bad Homburg, Germany) and used under conditions recommended by the manufacturers.Plasmids and bacterial strains. C. burnetii DNA fragments were cloned in phagemid vector pBluescript II KS(ϩ), cosmid vector SuperCos 1, or lambda phage vector EMBL4 (Stratagene). Recombinant cosmid and lambda phage DNAs were packaged (Gigapack II packaging extract; Stratagene) and transfected into Escherichia coli XL1 Blue (Stratagene). C. burnetii reference isolates Scurry Q217, Priscilla Q177, Dugway 7E22-...

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Section: Discussionmentioning

confidence: 99%

“…However, it was shown that Scurry Q217 contains chromosomally integrated plasmid-homologous DNA sequences (8). In an attempt to understand the significance of plasmids in C. burnetii, we examined plasmidless isolates with chromosomally integrated plasmid-homologous DNA fragments.…”

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confidence: 99%

Plasmid-homologous sequences in the chromosome of plasmidless Coxiella burnetii Scurry Q217

et al. 1997

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“…These plasmids range from 32 to 42 kb in size and share a common 25-kb "core" region along with unique regions (40,59,79). QpRS-like plasmid sequences are integrated into the chromosome of some isolates (40,60,79). Plasmid types are associated with specific genomic groups.…”

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confidence: 99%

Genetic Diversity of the Q Fever Agent,Coxiella burnetii, Assessed by Microarray-Based Whole-Genome Comparisons

Beare¹,

Samuel

Howe³

et al. 2006

J Bacteriol

125

132

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Coxiella burnetii, a gram-negative obligate intracellular bacterium, causes human Q fever and is considered a potential agent of bioterrorism. Distinct genomic groups of C. burnetii are revealed by restriction fragmentlength polymorphisms (RFLP). Here we comprehensively define the genetic diversity of C. burnetii by hybridizing the genomes of 20 RFLP-grouped and four ungrouped isolates from disparate sources to a high-density custom Affymetrix GeneChip containing all open reading frames (ORFs) of the Nine Mile phase I (NMI) reference isolate. We confirmed the relatedness of RFLP-grouped isolates and showed that two ungrouped isolates represent distinct genomic groups. Isolates contained up to 20 genomic polymorphisms consisting of 1 to 18 ORFs each. These were mostly complete ORF deletions, although partial deletions, point mutations, and insertions were also identified. A total of 139 chromosomal and plasmid ORFs were polymorphic among all C. burnetii isolates, representing ca. 7% of the NMI coding capacity. Approximately 67% of all deleted ORFs were hypothetical, while 9% were annotated in NMI as nonfunctional (e.g., frameshifted). The remaining deleted ORFs were associated with diverse cellular functions. The only deletions associated with isogenic NMI variants of attenuated virulence were previously described large deletions containing genes involved in lipopolysaccharide (LPS) biosynthesis, suggesting that these polymorphisms alone are responsible for the lower virulence of these variants. Interestingly, a variant of the Australia QD isolate producing truncated LPS had no detectable deletions, indicating LPS truncation can occur via small genetic changes. Our results provide new insight into the genetic diversity and virulence potential of Coxiella species.

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“…The cosmid pEST is a truncated version of pHK17 (9, 10) from which Tetr had been excised by a partial digestion with HaeII and then ligated with T4 ligase (22).…”

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Transformation of Rochalimaea quintana, a member of the family Rickettsiaceae

1990

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Rochalimaea quintana is the only member of the family Rickettsiaceae that can be grown in vitro. Because of its relationship to the other members of this family, techniques developed to transform R. quintana might be applicable to the obligate intracellular bacteria of the Rickettsiaceae. These procedures are critical to understanding mechanisms of pathogenesis and the nature of obligate intracellular growth. A transformation procedure for R. quintana has been established by using electroporation techniques. Several cosmids or plasmids with replicons RK2 and RSF1010 have been successfully used to transform this organism. Transformants were obtained by selection for antibiotic resistance to chloramphenicol or kanamycin. Plasmid retention and replication has been verified by Southern blot analysis and chloramphenicol acetyltransferase assay. Experimentation with different voltage field strengths and pulse times indicate that 12.5 kV/cm at 10 ms (25 ,LF and 400 fQ) was optimal, giving a transformation frequency of -0.3% and 3 x 105 transformants per ,ug of DNA.The family Rickettsiaceae contains a number of important human pathogens that are associated with arthropods. The members of this family differ markedly and have been separated into three genera: Rickettsia, Coxiella, and Rochalimaea (29).' Both Rochalimaea quintana and Rochalimaea vinsonii can be cultivated axenically and appear to grow in epicellular locations within their eucaryotic hosts (8,27). Members of this genus appear to be more closely related to some species of the genus Rickettsia than to Coxiella burnetii. Indeed, C. burnetii does not appear to be related to either Rickettsia or Rochalimaea species (24, 25). R. quintana has been shown to have metabolic and nutritional properties similar to Rickettsia spp. (27). In support of these findings, studies of the genetic relatedness of Rochalimaea and Rickettsia species by DNA-DNA hybridization analyses reveal a small but significant degree of hybridization (16). Recently, genetic analyses utilizing highly conserved rRNA sequences have confirmed the relationship between these two genera (26). Classical genetic studies of the genera Rickettsia and Coxiella have been severely restricted because of difficulties in manipulating these obligate intracellular organisms. The ability to transform these organisms would allow the introduction of specific mutations or genes and transformants could be examined for altered biological properties in vivo. It

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Comparison of Coxiella burnetii Plasmids to Homologous Chromosomal Sequences Present in a Plasmidless Endocarditis‐Causing Isolatea

Cited by 47 publications

References 14 publications

Plasmid-homologous sequences in the chromosome of plasmidless Coxiella burnetii Scurry Q217

Plasmid-homologous sequences in the chromosome of plasmidless Coxiella burnetii Scurry Q217

Genetic Diversity of the Q Fever Agent,Coxiella burnetii, Assessed by Microarray-Based Whole-Genome Comparisons

Transformation of Rochalimaea quintana, a member of the family Rickettsiaceae

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