Correction for Sandoz et al., Complementation of Arginine Auxotrophy for Genetic Transformation of Coxiella burnetii by Use of a Defined Axenic Medium

ABSTRACTWe recently described acidified citrate cysteine medium (ACCM), which supports host cell-free (axenic) growth ofCoxiella burnetii. After 6 days of incubation, greater than 3 logs of growth was achieved with the avirulent Nine Mile phase II (NMII) strain. Here, we describe modified ACCM and culture conditions that support improved growth ofC. burnetiiand their use in genetic transformation and pathogen isolation from tissue samples. ACCM was modified by replacing fetal bovine serum with methyl-β-cyclodextrin to generate ACCM-2. Cultivation of NMII in ACCM-2 with moderate shaking and in 2.5% oxygen yielded 4 to 5 logs of growth over 7 days. Similar growth was achieved with the virulent Nine Mile phase I and G isolates ofC. burnetii. Colonies that developed after 6 days of growth in ACCM-2 agarose were approximately 0.5 mm in diameter, roughly 5-fold larger than those formed in ACCM agarose. By electron microscopy, colonies consisted primarily of theC. burnetiismall cell variant morphological form. NMII was successfully cultured in ACCM-2 when medium was inoculated with as little as 10 genome equivalents contained in tissue homogenates from infected SCID mice. A completely axenicC. burnetiigenetic transformation system was developed using ACCM-2 that allowed isolation of transformants in about 2 1/2 weeks. Transformation experiments demonstrated clonal populations in colonies and a transformation frequency of approximately 5 × 10−5. Cultivation in ACCM-2 will accelerate development ofC. burnetiigenetic tools and provide a sensitive means of primary isolation of the pathogen from Q fever patients.

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Complementation of Arginine Auxotrophy for Genetic Transformation of Coxiella burnetii by Use of a Defined Axenic Medium

Applied and Environmental Microbiology ◽

10.1128/aem.00261-16 ◽

2016 ◽

Vol 82 (10) ◽

pp. 3042-3051 ◽

Cited By ~ 28

Author(s):

Kelsi M. Sandoz ◽

Paul A. Beare ◽

Diane C. Cockrell ◽

Robert A. Heinzen

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Transformation ◽

Host Cell ◽

Coxiella Burnetii ◽

Genetic Manipulation ◽

Defined Medium ◽

Gradual Transition ◽

Content Type ◽

Selection Of

ABSTRACTHost cell-free (axenic) culture ofCoxiella burnetiiin acidified citrate cysteine medium-2 (ACCM-2) has provided important opportunities for investigating the biology of this naturally obligate intracellular pathogen and enabled the development of tools for genetic manipulation. However, ACCM-2 has complex nutrient sources that preclude a detailed study of nutritional factors required forC. burnetiigrowth. Metabolic reconstruction ofC. burnetiipredicts that the bacterium cannot synthesize all amino acids and therefore must sequester some from the host. To examineC. burnetiiamino acid auxotrophies, we developed a nutritionally defined medium with known amino acid concentrations, termed ACCM-D. Compared to ACCM-2, ACCM-D supported longer logarithmic growth, a more gradual transition to stationary phase, and approximately 5- to 10-fold greater overall replication. Small-cell-variant morphological forms generated in ACCM-D also showed increased viability relative to that generated in ACCM-2. Lack of growth in amino acid-deficient formulations of ACCM-D revealedC. burnetiiauxotrophy for 11 amino acids, including arginine. Heterologous expression ofLegionella pneumophilaargGHinC. burnetiipermitted growth in ACCM-D missing arginine and supplemented with citrulline, thereby providing a nonantibiotic means of selection ofC. burnetiigenetic transformants. Consistent with bioinformatic predictions, the elimination of glucose did not impairC. burnetiireplication. Together, these results highlight the advantages of a nutritionally defined medium in investigations ofC. burnetiimetabolism and the development of genetic tools.IMPORTANCEHost cell-free growth and genetic manipulation ofCoxiella burnetiihave revolutionized research of this intracellular bacterial pathogen. Nonetheless, undefined components of growth medium have made studies ofC. burnetiiphysiology difficult and have precluded the development of selectable markers for genetic transformation based on nutritional deficiencies. Here, we describe a medium, containing only amino acids as the sole source of carbon and energy, which supports robust growth and improved viability ofC. burnetii. Growth studies confirmed thatC. burnetiicannot replicate in medium lacking arginine. However, genetic transformation of the bacterium with constructs containing the last two genes in theL. pneumophilaarginine biosynthesis pathway (argGH) allowed growth on defined medium missing arginine but supplemented with the arginine precursor citrulline. Our results advance the field by facilitating studies ofC. burnetiimetabolism and allowing non-antibiotic-based selection ofC. burnetiigenetic transformants, an important achievement considering that selectable makers based on antibiotic resistance are limited.

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A comparison of three cryotechniques (freeze-drying, cryosubstitution, cryosectioning) of specimen preparation for immunolabelling of bacterial antigens of Coxiella burnetii

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100168438 ◽

1994 ◽

Vol 52 ◽

pp. 140-141

Author(s):

T. F. McCaul ◽

R. J. Gould

Keyword(s):

Coxiella Burnetii ◽

Freeze Drying ◽

Stress Proteins ◽

Cultured Cells ◽

High Specificity ◽

Specimen Preparation ◽

Bacterial Antigens ◽

Structural Preservation ◽

Nuclear Ribonucleoprotein ◽

Cellular Components

Immuno-electron microscopy has allowed the selective localisation of molecules with high resolution and high specificity. Cryopreparatory methods have provided better retention of antigenicity suitable for precise immunolabelling together with optimal structural preservation of cellular components. Cryosubstitution and cryoultramicrotomy have widely been exploited for immunolabelling. Molecular Distillation Dryer (MDD), a form of freeze-drying technique, has recently been used for immunolabelling of Plasmodium falciparum stress proteins and nuclear ribonucleoprotein particles in cultured cells. In the present study, we report the comparison of all three cryotechniques in the immunolabelling of bacterial antigens of Coxiella burnetii.The highly infectious C. burnetii was prefixed in 3% glutaraldehyde (66 mM cacodylate buffer, pH 6.8 ). The cells were then pre-embedded in 2% low-temperature agarose on Durapore hydrophilic membrane prior to cryofixation using a LifeCell CF100 metal-mirror system. A 1% glutaraldehyde in 100% methanol was used as a medium for cryosubstitution in a Reichert CS Auto Cryosubstitution apparatus.

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