Despite the vast surface area of terrestrial plant leaves and the large microbial communities they support, little is known of the ability of leaf-associated microorganisms to access and degrade airborne pollutants. Here, we examined bacterial acquisition and degradation of phenol on leaves by an introduced phenol degrader and by natural phyllosphere communities. Whole-cell gfp-based Pseudomonas fluorescens bioreporter cells detected phenol on leaves that had previously been transiently exposed to gaseous phenol, indicating that leaves accumulated phenol; moreover, they accumulated it in sites that were accessible to epiphytic bacteria and to concentrations that were at least 10-fold higher than those in the air. After inoculated leaves were exposed to gaseous 14C-phenol, leaves harbouring the phenol-degrading Pseudomonas sp. strain CF600 released eight times more 14CO2 than did leaves harbouring a non-degrading mutant, demonstrating that CF600 actively mineralized phenol on leaves. We evaluated phenol degradation by natural microbial communities on green ash leaves that were collected from a field site rich in airborne organic pollutants. We found that significantly more phenol was mineralized by these leaves when the communities were present than by these leaves following surface sterilization. Thus, phenol-degrading organisms were present in these natural communities and were metabolically capable of phenol degradation. Collectively, these results provide the first direct evidence that bacteria on leaves can degrade an organic pollutant from the air, and indicate that bacteria on leaves could potentially contribute to the natural attenuation of organic air pollutants.

Microbial communities on aerial plant leaves may contribute to the degradation of organic air pollutants such as phenol. Epiphytic bacteria capable of phenol degradation were isolated from the leaves of green ash trees grown at a site rich in airborne pollutants. Bacteria from these communities were subjected, in parallel, to serial enrichments with increasing concentrations of phenol and to direct plating followed by a colony autoradiography screen in the presence of radiolabeled phenol. Ten isolates capable of phenol mineralization were identified. Based on 16S rDNA sequence analysis, these isolates included members of the genera Acinetobacter, Alcaligenes, and Rhodococcus. The sequences of the genes encoding the large subunit of a multicomponent phenol hydroxylase (mPH) in these isolates indicated that the mPHs of the gram-negative isolates belonged to a single kinetic class, and that is one with a moderate affinity for phenol; this affinity was consistent with the predicted phenol levels in the phyllosphere. PCR amplification of genes for catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) in combination with a functional assay for C23O activity provided evidence that the gram-negative strains had the C12O-, but not the C23O-, phenol catabolic pathway. Similarly, the Rhodococcus isolates lacked C23O activity, although consensus primers to the C12O and C23O genes of Rhodococcus could not be identified. Collectively, these results demonstrate that these leaf surface communities contained several taxonomically distinct phenol-degrading bacteria that exhibited diversity in their mPH genes but little diversity in the catabolic pathways they employ for phenol degradation.

Chapter 3. Identification and genetic characterization of phenol-degrading bacteria from leaf microbial communities..