Diversity and evolution of the phenazine biosynthesis pathway

Abstract

Phenazines are versatile secondary metabolites of bacterial origin that function in biological control of plant pathogens and contribute to the ecological fitness and pathogenicity of the producing strains. In this study, we employed a collection of 94 strains having various geographic, environmental, and clinical origins to study the distribution and evolution of phenazine genes in members of the genera Pseudomonas, Burkholderia, Pectobacterium, Brevibacterium, and Streptomyces. Our results confirmed the diversity of phenazine producers and revealed that most of them appear to be soil-dwelling and/or plant-associated species. Genome analyses and comparisons of phylogenies inferred from sequences of the key phenazine biosynthesis (phzF) and housekeeping (rrs, recA, rpoB, atpD, and gyrB) genes revealed that the evolution and dispersal of phenazine genes are driven by mechanisms ranging from conservation in Pseudomonas spp. to horizontal gene transfer in Burkholderia spp. and Pectobacterium spp. DNA extracted from cereal crop rhizospheres and screened for the presence of phzF contained sequences consistent with the presence of a diverse population of phenazine producers in commercial farm fields located in central Washington state, which provided the first evidence of United States soils enriched in indigenous phenazine-producing bacteria.

FIG. 1.

Comparison of neighbor-joining phylogenies inferred from data for aligned 391-bp fragments of phzF (A) and 1,440-bp fragments of rrs (B) of Pseudomonas spp. In addition to sequences from phenazine-producing strains, the data set used to infer the 16S rRNA gene phylogeny included sequences from type strains of the P. chlororaphis (PC), P. fluorescens (PF), P. syringae (PSy), P. putida (PP), P. aeruginosa (PA), and P. stutzeri (PSt) species complexes as defined by Anzai et al. (4). Indels were ignored in the analysis, and evolutionary distances were estimated using the Kimura two-parameter model of nucleotide substitution. Bootstrap values greater than 60% are indicated at the nodes, and scale bars indicate substitutions per site. The branch lengths are proportional to the amount of evolutionary change. Sequences of phzF retrieved from the GenBank database and sequences generated in this study are indicated by open and filled circles, respectively. Phenazine-producing species used for further phylogenetic analyses are indicated by bold type.

FIG. 2.

Neighbor-joining phylogenies inferred from data for rrs sequences of Burkholderia spp. (A) and recA genes of the B. cepacia species complex (B). Indels were ignored, and the data sets used for analysis of rrs and recA contained 1,307 and 561 characters, respectively. Evolutionary distances were estimated using the Kimura two-parameter model of nucleotide substitution. Bootstrap values greater than 60% are indicated at the nodes, and the scale bar indicates 0.005 substitution per site. The branch lengths are proportional to the amount of evolutionary change. Phenazine-producing strains are indicated by bold type. Open circles indicate species whose genomes have been sequenced.

FIG. 3.

Comparison of neighbor-joining phylogenies inferred from data for phzF (A) and concatenated housekeeping genes (rrs, recA, rpoB, atpD, and gyrB) (B) of the core set of phenazine-producing species. Indels were ignored in the analysis, and the phzF and concatenated housekeeping gene data sets contained 378 and 4,286 characters, respectively. Evolutionary distances were estimated using the Kimura two-parameter model of nucleotide substitution. The reproducibility of clades was assessed by bootstrap resampling with 1,000 pseudoreplicates, and bootstrap values greater than 60% are indicated at the nodes. The branch lengths are proportional to the amount of evolutionary change. The scale bars indicate substitution per site. Nodes with nearly identical sequences were collapsed. The phzF alleles originating from rhizosphere DNA are indicated by solid diamonds followed by a digit corresponding to the number of sequences in the collapsed node followed by a letter(s) indicating the crop (SW, spring wheat; WW, winter wheat; B, barley; AA, alfalfa) from which rhizosphere DNA was extracted. Shading indicates sequences that did not cluster similarly in the two trees. Bold type indicates organisms that were used for maximum likelihood and Bayesian analyses.

FIG. 4.

Organization of the phenazine biosynthesis loci in different bacterial species (A) and physical maps of putative composite phenazine transposons from Burkholderia spp. (B). Genes and their orientations are indicated by arrows. Homologous genes in P. fluorescens 2-79 (44), P. chlororaphis 30-84 (44, 50), P. chlororaphis PCL1391 (14), Pseudomonas sp. CMR12a (M. Höfte, personal communication), P. aeruginosa PAO1 (59), P. atrosepticum SCRI1043 (7), P. agglomerans Eh1087 (21), B. lata 383, B. glumae BGR-1, B. linens BL2, N. dassonvillei subsp. dassonvillei DSM43111, S. cinnamonensis DMS1042 (24), and S. anulatus LU9663 (55) are indicated by arrows that are the same color, whereas unique species-specific genes are indicated by open arrows. For sequenced genomes, locus tags are indicated using a code; e.g., the locus tag for the phzB homologue of B. lata 383 is Bcep18194_B1568. In panel A, the sizes of genes and intergenic regions are not to scale, and the arrows with bold outlines indicate known or putative phenazine transport genes. In panel B, the open arrows represent intact and inactive (indicated by asterisks) transposase genes, and the rectangles indicate putative left (LE) and right (RE) transposon ends.