Cloning experiments MAPK inhibitor were conducted using the pGEM-T® Easy vector (Promega). Ligated products were transformed into Escherichia coli TOP10 competent cells, and positive transformants were color-screened on LB plates supplemented with ampicillin (100 μg mL−1), X-Gal (80 μg mL−1), and isopropyl-beta-d-thiogalactopyranoside (IPTG 0.5 mM). Clones were selected using primers M13F-20 and M13R and selected according to the expected size (620 bp) of the amplified LmPH gene fragment. Positive PCR products of the expected size
were sequenced using the vector-specific primer M13F-20 at the Macrogen service (Macrogen, Seoul, South Korea). Sequences were manually refined using the BioEdit package. Amino acid-derived sequences were further aligned using
clustalw. Amino acid-derived sequence alignments of partial LmPH were used to construct a distance matrix using the online package implemented in mothur v1.13 (Schloss et al., 2009). Rarefaction curves were calculated at a cutoff value of 90% similarity and were used to determine the number of operational taxonomic units (OTUs) in each sample. A 90% cutoff value of the LmPH gene approaches a species-level OTU definition according to comparisons between available 16S rRNA and LmPH gene sequences of cultured phenol oxidizers (results not shown). Estimated richness (SChao, and SAce), Shannon diversity index (H′), and evenness (E′) indices were calculated according to the OTUs find more distribution. Jaccard similarity coefficients were calculated pairwise by using either the presence of shared OTUs between two different communities (OTU based approach) or the relative abundance of individuals that belong to shared OTUs (abundance-based test). Phylogeny was reconstructed using mega v.4. The Amino Poisson correction and pairwise deletion methods were used. Bootstrap analysis was conducted with 1000 replications. Additionally, to estimate the diversity between different bacterial communities using
the phylogenetic information, UniFrac (UniFrac weighted PFKL algorithm) and parsimony tests were calculated using the above phylogenetic tree. The outcomes of these analyses reflect the evolutionary distance between the members of the analyzed bacterial communities (Lozupone et al., 2011). LmPH sequences obtained in this study have been submitted to GenBank under accession numbers JF806548–JF806617 and JQ069975–JQ070053. During the duration of the whole experiment (112 days), a significant relationship between leaf bacterial biomass and phenol oxidase activity was observed, suggesting a link between bacteria and degradation of phenols in leaves (Fig. 1). To investigate the potential role of phenol-degrading bacteria, three dates were selected for molecular analysis of the largest subunit of multicomponent phenol hydroxylases (LmPHs).