The first steps in developing Pseudomonas sp. (CT 364) involve identifying antibiotics active that it is susceptible to in order to select transformants and optimistaion of transformation protocols. Five antibiotics were tested and two were found to be active against Pseudomonas sp. Working concentrations were identified for each antibiotic using lab automation. Additionally, a new streptomycin resistance cassette was constructed to be used in building plasmids for Pseudomonas sp.

Pseudomonas sp. (CT 364) was obtained from DSMZ, Germany (DSM No.: 25356). The strain arrived freeze-dried and was revived according to the protocol recommended by DSMZ.(Figure 1)

Figure 1. Pseudomonas sp. DSM 25356 plated on tryptone soy agar

Screening on tryptone soy agar (TSA) showed Pseudomonas sp. to be resistant to chloramphenicol, kanamycin and carbenicillin. Antibiotic concentrations of 50 and 100 µg/ml were tested with lawns forming on agar containing 100 µg/ml of each antibiotic. (Figures 2, 3 & 4) after 24 hours incubation at 28 °C.

Figure 2. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing chloramphenicol (100 µg/ml)

Figure 3. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing carbenicillin (100 µg/ml)

Figure 4. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing carbenicillin (100 µg/ml)

Screening on TSA showed that Pseudomonas sp. was susceptible to both streptomycin (Figure 5) and gentamicin (Figure 6) with no colony forming units (CFUs) visible on agar containing 50 µg/ml of either antibiotic after 24 hours incubation at 28 °C.

Figure 5. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing streptomycin (100 µg/ml)

Figure 6. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing gentamicin (100 µg/ml)

After identifying which antibiotics were active against Pseudomonas sp. The next step was to identify working concentrations of these antibiotics to be used when selecting transformants. This was done by carrying out minimum inhibitory concentration (MIC) experiments where growth was tested against a range of antibiotic concentrations.

The results of our MIC experiments showed a clear dose response between antibiotic concentration and growth of Pseudomonas sp. for both streptomycin and gentamicin (Figures 7 and 8). Gentamicin was found to be the more effective antibiotic with a concentration of 1.5 µg/ml sufficient to prevent growth. A concentration of 6.0 µg/ml of streptomycin was required to prevent growth. A slight increase in absorbance was observed for the positive control for both antibiotics. This is likely due to release of compounds by bacterial cells upon death.

Figure 7. Pseudomonas sp. DSM 25356 grown in tryptone soy broth containing gentamicin at varying concentrations. Cells were grown in 96-well plate format in 200 µl volumes at 37 °C over 24 hours. (n=4 replicates, error bars are standard error of the mean)

Figure 8. Pseudomonas sp. DSM 25356 grown in tryptone soy broth containing streptomycin at varying concentrations. Cells were grown in 96-well plate format in 200 µl volumes at 37 °C over 24 hours. (n=4 replicates, error bars are standard error of the mean).

After identifying the MIC in liquid culture, the MICs in agar were determined for gentamicin as this was the antibiotic being taken forward for transformation.

Figure 8. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing gentamicin (4 µg/ml)

Figure 9. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing gentamicin (6 µg/ml)

Agar assays showed that concentrations of 2 µg/ml and 4 µg/ml of antibiotic were insufficient to prevent growth of Pseudomonas sp. on agar with a lawn forming on 2 µg/ml and some colonies forming on 4 µg/ml (Figure 7). A gentamicin concentration of 6 µg/ml was sufficient to prevent growth of Pseudomonas sp. as no colonies formed at this concentration (Figure 8).