Phenazine antibiotic production in liquid culture, on surface agar, and on plant roots by Pseudomonas aureofaciens PGS12
The growth of P. aureofaciens PGS12 was followed in nutrient broth, on nutrient agar, and on plant roots by monitoring cell numbers, the production of the auto inducer N-acyl- L-hexanoyl homo serine lactone (HHL), and the antibiotic phenazine-l-carboxylic acid (PCA). In nutrient broth, as the growth rate declined, HHL synthesis increased rapidly. Up to 38% more HHL was produced compared to the increase in cell numbers indicating that transcription of the phenazine operon was auto induced. As the bacterial culture entered stationary phase, HHL concentration declined rapidly while PCA concentration continued to accumulate at a high rate. In stationary phase, HHL concentration continued to decline while PCA accumulated slowly. A promoterless mini-Tn5-luxAB transposon was used to generate isogenic strains of P. aureofaciens PGSI2. Strain BI03 was shown to have the luxAB reporter gene inserted in the phzB gene within the phenazine operon. Phenazine transcriptional activity (bioluminescence) was compared with the light output from a constitutive reporter, strain 117. Levels and pattern of bioluminescence from strain B 1 03 followed closely HHL production and indicated that gene expression was maximal in transition phase and silenced in stationary phase. PCA production continued in stationary phase suggesting that the protein products of the phenazine operon were maintained in the cell after down regulation. The induction of the phenazine operon started in nutrient broth when cell density was ca. 2x 108 cells mrl and HHL had accumulated to a threshold concentration of 0.63x 1 0- 6 ± O.3x 1 0- 6 ng celrl. ± On NA, cells were in a transition phase of growth for at least 9 h. The cell density was 55 to 75 times higher within a colony than in liquid culture. The maximal production of HHL and light output per calculated equivalent volumes were also between 50 and 65 times higher on NA than in broth. The maximal light output and maximal HHL accumulation per cell were similar on both media. Therefore, the increased levels on NA may be mainly due to a higher cell number in the colony. The production of the antibiotic PCA per cell was ca. 7 times higher in a colony than in NB, and the production in an equivalent volume was ca. 360 times higher in a colony. Therefore, the higher PCA concentration in the colony cannot be explained solely on the basis of an increase in cell density. The auto inducer concentration remained high within a colony for a prolonged period of time compared to the burst seen in NB. A high concentration of HHL per cell for a longer period of time may have sustained the greater production and accumulation of PCA in the colony. Similarly the transcriptional activity of the phenazine operon, as reflected by phzB::luxAB expression in strain BI03, remained maximal during this time. In contrast to laboratory culture studies, in all the experiments where P. aureofaciens PGS 12 was inoculated onto roots, neither HHL nor PCA was detected, although the bacteria colonised the bean roots and wheat seedlings efficiently. On wheat seedlings, the transcriptional activity from both reporter strains decreased during the experiment. On bean roots, bioluminescence per cell from strains 117 and B 1 03 increased 25-fold during the first 3 days and the ratio of bioluminescence from strains B 1 03 over 117 indicated an up to 5 times greater transcriptional activity from strain B 1 03 than from strain 117. The minimum levels for the detection of HHL and PCA were low. Therefore, either these compounds were produced in minute amounts, or HHL and phenazine were degraded or adsorbed onto the plant material lowering their levels below the detection limit.