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Detection of sequences related to biosynthesis of antifungal metabolites and identification of a PhzF-like gene in a Pseudomonas rhizobacterium of Piper tuberculatum

Author(s): O.S. Silva Jr., N.L.F. Barros, A.S. Siqueira, E.C. Gonçalves, D. N. Marques, S.P. Reis, and C.R.B. Souza

Our previous studies identified a Pseudomonas putida (Pt12 isolate) associated with roots of Piper tuberculatum Jacq., a Piperaceae occurring in the Amazon region with known resistance to infection by Fusarium solani f. sp. piperis, the causal agent of root rot disease in black pepper (Piper nigrum L.). This Pt12 isolate was able to inhibit in vitro growth of this fungus 39%. However, the inhibition mechanisms were unknown. Here, we searched for genes coding enzymes involved in the biosynthesis of compounds with potential antifungal activity in the Pt12 isolate, such as phenazine, pyoluteorin, 2,4-diacetylphloroglucinol and hydrogen cyanide. We found sequences potentially related to biosynthesis of phenazine (PhzF) based on DNA sequencing and comparative sequence analyses using the GenBank DataBase. PCR and nested PCR assays were used to isolate a 789-bp sequence coding for a deduced protein with 262 amino acid residues with high identity with a PhzF family phenazine biosynthesis protein from P. putida (WP_046785327.1), P. plecoglossicida (WP_041505738.1) and P. parafulva (WP_039578870.1). Molecular modeling and molecular dynamic simulations revealed that putative PhzF of Pt12 isolate could stably interact with substrate DHHA (Trans-2, 3-dihydro-3- hydroxyanthranilic acid) through some amino acid residues that are similar to conserved amino acids critical to the catalytic activity of a well-characterized PhzF protein produced by Pseudomonas fluorescens.