Effect of biofilm and the role of rpoS on the survival of a p-Nitrophenol-degrading Pseudomonas putida / by Miranda Maki.
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Pseudomonas putida is a Gram negative bacterium that can be found naturally in the environment as planktonic free-living cells, or in concert with other bacterial species in biofilms. In this study, the P. putida strain has the ability to degrade p -nitrophenol (PNP), a moderately toxic organic pollutant found world-wide. Both the survivability and PNP degradative abilities of planktonic and biofilm P. putida cells were compared in buffer and river water samples containing varying concentrations of PNP. The P. putida biofilm cells showed higher survival rate in both the buffer and river water samples compared to the planktonic P. putida cells. In the buffer experiment, P. putida planktonic cell density decreased from 8.25 to 5.7 and 5.0 log (CFU/mL) after 17 days treatment with 3000 and 4000 μM PNP. However, P. putida biofilm cells in the buffer system had a decrease in cell density from 10.2 to 8.7 and 9.0 log (CFU/g glass wool) after 17 days treatment with 3000 and 4000 μM PNP, respectively. The survival fraction revealed that biofilm cells had a 100-fold greater survival compared to planktonic cells at 3000 μM and 4000 μM PNP by day 17. In the river water system at 3000 and 4000 μM PNP, the planktonic cell density decreased from 9.4 to 3.0 log (MPN/mL) and biofilm cell density decreased from 10.3 to 9.5 and 10 log (MPN/g glass wool), respectively, after 17 days treatment. Survival fractions of the bacteria in the river water system revealed that biofilm cells survived 350- and 450-fold greater than planktonic cells at 3000 and 4000 μM PNP, respectively. Monitoring PNP degradation in the buffer system, revealed that the P. putida planktonic cells degraded 1500 μM PNP completely, degraded 2600 μM PNP by 50% and 3000 μM PNP by 40% and did not degrade 4000 μM PNP within 18 days. The biofilm cells in the buffer completely degraded 1500 and 2600 μM PNP, degraded 3000 μM PNP by 55%, and did not degrade 4000 μM PNP, in 18 days. In the river water system, the P. putida planktonic cells did not degrade any PNP. By contrast, the P. putida biofilm cells in the river water were found to completely degrade 1500 μM PNP in 9 days. It was observed that high concentrations of PNP inflicted injury to cells located on the surface of biofilms while cells in the interior of biofilms remained viable. The P. putida biofilms were able to survive in and degrade PNP better than planktonic cells, and these findings indicate potential improvement of bioremediation using biofilm cells. The expression of the stationary phase sigma factor gene (rpoS) and biofilm formation are important bacterial stress-survival strategies. To further explore the relationship between these two factors, the survival and competitiveness of the PNP-degrading P. putida strain as well as its rpoS mutant were examined in both planktonic and biofilm phases. To distinguish wild-type (WT) and knock-out (KO) strains in mixed samples, they were labeled with a red fluorescent protein-gentamycin resistant and a green fluorescent protein-gentamycin resistant gene cassette via a Tn7 transposon system, respectively. In single cultures, the KO-gfp biofilm featured a greater than 4-fold increase in biovolume than the WT-rfp. However, mixed biofilm biovolume revealed that KO-gfp was reduced by 75.6% compared to the single culture KO -gfp biofilm biovolume, suggesting that the WT-rfp suppressed KO-gfp biofilm formation. Competitiveness studies showed that carbon-starved planktonic WT-rfp single cultures achieved a 3.5-fold greater survival rate in 0.85% saline than their KO-gfp counterparts, and out-competed them by more than 13-fold in mixed culture samples. Conversely, there were no significant survival differences between the KO-gfp and WT-rfp strains in biofilm samples. Finally, differences in cellular cohesiveness were evident, after 60 min washing with 0.2% SDS resulted in 32 and 89% cells detached from the KO and WT samples, respectively. These results indicate that although the KO produced more biofilm than the WT, it may in fact not confer an advantage in survival because the KO biofilm has a lower percentage of surviving cells and cannot readily detach to colonize new habitats. Overall, these findings provide new perspectives towards a more complete understanding of the role of rpoS in biofilm formation.