Detailed Study Notes on Nonchromosomal Antibiotic Resistance in Bacteria

Stanley N. Cohen, Annie C. Y. Chang, and Leslie Hsu
- Division of Clinical Pharmacology, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305
- Communicated by A. D. Kaiser, May 16, 1972

Main Finding: Transformation of E. coli cells treated with Calcium Chloride (CaCl2) resulted in multiple antibiotic resistance by purified R-factor DNA.
Transformational Mechanics:
- Drug resistance begins to manifest in a small fraction of recipient bacterial population shortly after DNA uptake.
- Complete genetic expression of resistance necessitates a subsequent incubation in drug-free medium before antibiotic challenge.
Characteristics of Transformed DNA: The transformed bacteria gain a closed circular, transferable DNA species retaining the resistance, fertility, and sedimentation characteristics of the parent R factor.
Effective Forms of R-factor DNA:
- Covalently-closed, catenated, and nicked circular forms of R-factor DNA are effective in transformation.
- Denaturation and sonication negate the transforming ability of R-factor DNA.

R factors in Enterobacteriaceae are autonomously replicating units of extrachromosomal DNA.
Certain R factors consist of reversible covalent linkage of separate plasmids carrying distinct resistance or transfer functions.
Electron microscopy analyses corroborate previous findings about the molecular nature and structure of R factors.
Fundamental questions persist regarding gene interplay located in different regions of R-factor DNA and its roles in replication, genetic expression, recombination, and transfer.

Development of a genetic transformation system using purified molecular species of R-factor DNA enables the study of critical aspects of R-factor biology.
Transformation of E. coli has been extensively explored in other species, such as Pneumococcus and Bacillus subtilis, but largely ineffective for E. coli until recent advancements.

R factors identified:
- R64-11: Specifies resistance to tetracycline (Tc) and streptomycin (Sm). [ Source: R. Curtiss ]
- R6: Carries resistance to kanamycin (Km), neomycin (Nm), chloramphenicol (Cm), sulphonamide (Su), and tetracycline (Tc). [ Source: T. Watanabe ]
- R6-5: Variant of R6, without tetracycline resistance. [ Independently isolated in the laboratory ]

Techniques used:
- Covalently-closed R-factor DNA isolation was performed as described in references (6, 7).
- Alternative Brij-lysis technique for initial sample isolation.
- Purification via cesium chloride-ethidium bromide gradients.
- Fragments were obtained by sonication and denaturation at specific conditions.

**Cultivation of *E. coli*:
- Strain C600 grown at 37°C in media to reach an optical density of 0.85 at 590 nm.
Preparation for Transformation:
- Quick chilling of cells followed by sedimentation and washing in 10 mM NaCl.
- Resuspension in chilled CaCl2 for transformation capability enhancement.
DNA Incubation:
- DNA mixed with competent cells at specific concentrations followed by incubation at low temperature.
Heat Shock Treatment:
- Application of a 2-minute heat pulse at 42°C to facilitate DNA uptake.
Culturing the Transformed Cells:
- Plated on nutrient agar or diluted in L broth for incubation at 37°C.

Drugs Used:
- Neomycin: 25 µg/ml
- Streptomycin: 10 µg/ml
- Tetracycline: 25 µg/ml
- Kanamycin: 25 µg/ml
- Chloramphenicol: 25 µg/ml

Kanamycin Resistance: Expressed rapidly post DNA uptake; increases up to 1000-fold during subsequent incubation in drug-free medium, plateau reached in about 1 hour.

Findings: Transformation correlated linearly with R-factor DNA concentration.
Approximately 10^5 transformed bacteria per µg of R-factor DNA noted.

Importance of DNA Form: Closed, circular, catenated, and open-circular R-factor DNA demonstrated effective transformation.
Impact of Denaturation/Sonication: Both destroy transforming activity, as does prior DNase treatment.
The phenomenon observed: The DNA of the R factor R64-11 showed lower efficiency compared to R6-5 in transformations.

Observed Transformants:
- Verified across various antibiotics (km, nm, cm, tc), but no transformants pertinent to streptomycin on initial selection, indicating potential delayed expression requiring further incubation.

Isolation followed by cesium chloride gradients revealed closed circular DNA indistinguishable from parent R factor.
R-factor DNA can be transferred by conjugation at effective frequencies similarly to R factor's natural conjugation profile.

Purified R-factor DNA serves as an effective means to transform E. coli into multidrug-resistant cells rapidly.
Resulting antibiotic resistance can be the basis for deeper studies into plasmid function and bacterial resistance mechanisms.
The observations also establish groundwork for potential exploration of nontransferable independently replicating plasmids.

Thanks to Peter Lobban and Terrie Masuda for contributions regarding the technique of the calcium chloride transformation assay.
Supported by Grant AI 08619 from the National Institute of Allergy and Infectious Diseases and NSG GB 30581 from the National Science Foundation.

Citations ranging from Anderson (1968) to Davies et al. (1971) detailing foundational research in microbiology and R factor studies relevant to antibiotic resistance in bacteria.