Distribution of 2,4-Diacetylphloroglucinol Biosynthetic Genes among the Pseudomonas spp. Reveals Unexpected Polyphyletism

Abstract

Fluorescent pseudomonads protecting plant roots from phytopathogens by producing 2,4-diacetylphloroglucinol (DAPG) are considered to form a monophyletic lineage comprised of DAPG⁺Pseudomonas strains in the "P. corrugata" and "P. protegens" subgroups of the "Pseudomonas fluorescens" group. However, DAPG production ability has not been investigated for many species of these two subgroups, and whether or not the DAPG⁺Pseudomonas are truly monophyletic remained to be verified. Thus, the distribution of the DAPG biosynthetic operon (phlACBD genes) in the Pseudomonas spp. was investigated in sequenced genomes and type strains. Results showed that the DAPG⁺Pseudomonas include species of the "P. fluorescens" group, i.e., P. protegens, P. brassicacearum, P. kilonensis, and P. thivervalensis, as expected, as well as P. gingeri in which it had not been documented. Surprisingly, they also include bacteria outside the "P. fluorescens" group, as exemplified by Pseudomonas sp. OT69, and even two Betaproteobacteria genera. The phl operon-based phylogenetic tree was substantially congruent with the one inferred from concatenated housekeeping genes rpoB, gyrB, and rrs. Contrariwise to current supposition, ancestral character reconstructions favored multiple independent acquisitions rather that one ancestral event followed by vertical inheritance. Indeed, based on synteny analyses, these acquisitions appeared to vary according to the Pseudomonas subgroup and even the phylogenetic groups within the subgroups. In conclusion, our study shows that the phl⁺Pseudomonas populations form a polyphyletic group and suggests that DAPG biosynthesis might not be restricted to this genus. This is important to consider when assessing the ecological significance of phl⁺ bacterial populations in rhizosphere ecosystems.

Keywords: 2,4-diacetylphloroglucinol; DAPG; biocontrol agent; phl Operon; phlACBD genes.

Figure 1

Ancestral state character reconstruction by Maximum Parsimony of the gains and losses of the phl operon. (A) Phylogenetic rrs-gyrB-rpoB tree of selected Pseudomonas strains in which each node indicates presence (black circle) or absence of phl operon (phlACBD) (white circle). The red circle indicates multiple, equally-parsimonious scenarios that are shown in (B,C). The outer ring represents the subgroups defined in the “Pseudomonas fluorescens” group according to Mulet et al. (2010), the second ring correspond to the presence (black square) or the absence (white square) of the phl cluster. The different Pseudomonas groups are highlighted in color: Gray color, “P. chlororaphis” group; Green, “P. syringae” group; Purple, “P. putida” group; Yellow, “P. oleovorans” group; Red, “P. stutzeri” group; Blue, “P. aeruginosa” group. Fluorescent Pseudomonas strains of uncertain taxonomic status are written as “sp.” and those misclassified were renamed based on rrs-rpoD-gyrB phylogeny and ANI data (See Tables S2–S7). (B,C) The two equally parsimonious scenarios inferred by ancestral reconstruction for the most recent common ancestor of the “P. corrugata” subgroup. Contradicting nodes are denoted by a red outer circle. Strains P97.30, Q37-87, and Q65c-80 likely belong to P. brassicacearum but are portrayed as Pseudomonas sp. in the absence of ANI data.

Figure 2

Synteny of the genomic regions flanking the phl cluster among Pseudomonas genomes. The phl cluster is colored in red. Letters correspond to the name of each phl gene. Type strains are shown in bold, and letters following the strain name correspond to the multilocus phylogenetic groups defined in Frapolli et al. (2007). Each group of genes of homologous synteny downstream (at the left of the phl cluster) or upstream (at the right of the phl cluster) is presented using the same color/pattern, and strain-specific genes using white color. Brackets indicate the beginning or the end of a contig. The unclassified strains are positioned in a subgroup according to the phylogeny obtained in Figure 1. Fluorescent Pseudomonas strains of uncertain taxonomic status are written as “sp.” and those misclassified were renamed based on rrs-rpoD-gyrB phylogeny and ANI data (See Tables S2–S7).