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, 188 (7), 2309-24

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

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

Paul A Beare et al. J Bacteriol.

Abstract

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 fragment-length 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.

Figures

FIG.1.
FIG.1.
Comparison of the genomes of 22 C. burnetii isolates with the genome of the Nine Mile (RSA493) reference isolate. Labeled DNA was hybridized to the RMLchip_a containing probe sets specific for all ORFs of the Nine Mile isolate. ORF polymorphisms were identified as ORF probe sets showing log2 hybridization ratios lower than −1 that were validated as described in Materials and Methods. Red ORFs were completely deleted, blue ORFs were partially deleted, and yellow ORFs contained point mutation(s) or small insertions. The number and location of each polymorphism, consisting of single or multiple ORFs, is shown on the right. (A) Chromosomal polymorphisms 1 through 44. (B) Plasmid polymorphisms 45 through 51. The number and location of each polymorphism, consisting of either single or multiple ORFs, is shown on the right. Predicted nonfunctional NMI ORFs containing point mutations (PM) or frameshift (FS) mutations are indicated.
FIG.1.
FIG.1.
Comparison of the genomes of 22 C. burnetii isolates with the genome of the Nine Mile (RSA493) reference isolate. Labeled DNA was hybridized to the RMLchip_a containing probe sets specific for all ORFs of the Nine Mile isolate. ORF polymorphisms were identified as ORF probe sets showing log2 hybridization ratios lower than −1 that were validated as described in Materials and Methods. Red ORFs were completely deleted, blue ORFs were partially deleted, and yellow ORFs contained point mutation(s) or small insertions. The number and location of each polymorphism, consisting of single or multiple ORFs, is shown on the right. (A) Chromosomal polymorphisms 1 through 44. (B) Plasmid polymorphisms 45 through 51. The number and location of each polymorphism, consisting of either single or multiple ORFs, is shown on the right. Predicted nonfunctional NMI ORFs containing point mutations (PM) or frameshift (FS) mutations are indicated.
FIG.1.
FIG.1.
Comparison of the genomes of 22 C. burnetii isolates with the genome of the Nine Mile (RSA493) reference isolate. Labeled DNA was hybridized to the RMLchip_a containing probe sets specific for all ORFs of the Nine Mile isolate. ORF polymorphisms were identified as ORF probe sets showing log2 hybridization ratios lower than −1 that were validated as described in Materials and Methods. Red ORFs were completely deleted, blue ORFs were partially deleted, and yellow ORFs contained point mutation(s) or small insertions. The number and location of each polymorphism, consisting of single or multiple ORFs, is shown on the right. (A) Chromosomal polymorphisms 1 through 44. (B) Plasmid polymorphisms 45 through 51. The number and location of each polymorphism, consisting of either single or multiple ORFs, is shown on the right. Predicted nonfunctional NMI ORFs containing point mutations (PM) or frameshift (FS) mutations are indicated.
FIG. 2.
FIG. 2.
LPS profiles and ORF polymorphisms of C. burnetii isolates synthesizing different LPS chemotypes. (A) Silver-stained sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis profiles of purified LPS molecules from C. burnetii Nine Mile I (NMI), Nine Mile II (NMII), Nine Mile Crazy (NMC), and Australia QD (Au) isolates. The relative sizes of molecular mass markers in kilodaltons are shown on the left. (B) Comparison of the genomes of NMII, NMC, and Au isolates with the genome of the Nine Mile (RSA493) reference isolate. Labeled DNA was hybridized to the RMLchip_a containing probe sets specific for all ORFs of the Nine Mile isolate. ORF polymorphisms were identified as ORF probe sets showing log2 hybridization ratios lower than −1. Red ORFs were completely deleted and blue ORFs were partially deleted. Partial ORFs that were previously identified by Hoover at al. (35), but not detected by the CGH, are depicted in green.
FIG. 3.
FIG. 3.
Parsimony analysis of chromosomal and plasmid ORF content of C. burnetii isolates. Phylogenetic trees were constructed by using both chromosomal and plasmid ORFs (with 123 of 132 variable characters being parsimony informative and equally weighted) (A), chromosomal ORFs (with 93 of 102 variable characters being parsimony informative and equally weighted) (B), or plasmid ORFs (with all 30 variable characters being parsimony informative and equally weighted) (C). One of the two most parsimonious trees for each of these groups is depicted. (See Fig. S2 in the supplemental material for the alternative tree.) Proposed genomic groups (I to VIII) are shown in panel A. The horizontal bar indicates the number of character changes.

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