Functional Analysis of Phenazine Biosynthesis Genes in Burkholderia spp

Abstract

Burkholderia encompasses a group of ubiquitous Gram-negative bacteria that includes numerous saprophytes as well as species that cause infections in animals, immunocompromised patients, and plants. Some species of Burkholderia produce colored, redox-active secondary metabolites called phenazines. Phenazines contribute to competitiveness, biofilm formation, and virulence in the opportunistic pathogen Pseudomonas aeruginosa, but knowledge of their diversity, biosynthesis, and biological functions in Burkholderia is lacking. In this study, we screened publicly accessible genome sequence databases and identified phenazine biosynthesis genes in multiple strains of the Burkholderia cepacia complex, some isolates of the B. pseudomallei clade, and the plant pathogen B. glumae We then focused on B. lata ATCC 17760 to reveal the organization and function of genes involved in the production of dimethyl 4,9-dihydroxy-1,6-phenazinedicarboxylate. Using a combination of isogenic mutants and plasmids carrying different segments of the phz locus, we characterized three novel genes involved in the modification of the phenazine tricycle. Our functional studies revealed a connection between the presence and amount of phenazines and the dynamics of biofilm growth in flow cell and static experimental systems but at the same time failed to link the production of phenazines with the capacity of Burkholderia to kill fruit flies and rot onions.IMPORTANCE Although the production of phenazines in Burkholderia was first reported almost 70 years ago, the role these metabolites play in the biology of these economically important microorganisms remains poorly understood. Our results revealed that the phenazine biosynthetic pathway in Burkholderia has a complex evolutionary history, which likely involved horizontal gene transfers among several distantly related groups of organisms. The contribution of phenazines to the formation of biofilms suggests that Burkholderia, like fluorescent pseudomonads, may benefit from the unique redox-cycling properties of these versatile secondary metabolites.

Keywords: Burkholderia; biosynthesis; phenazine.

FIG 1

Multilocus sequence typing phylogeny of Burkholderia spp. Neighbor-joining phylogeny inferred from concatenated protein sequences (4,458 characters) of housekeeping enzymes AtpD, GltB, GyrB, RecA, LepA, PhaC, and TrpB. Sequences from P. phenazinium LMG 2247^T were used as an outgroup. Strains carrying phenazine biosynthesis genes are highlighted in bold font. Indels were ignored in the analysis, and evolutionary distances were estimated using the Jukes-Cantor genetic distance model. The reproducibility of clades was assessed by bootstrap resampling, and bootstrap values greater than 60% are indicated by gray circles at the nodes (circle sizes are proportional to bootstrap values). The branch lengths are proportional to the amount of evolutionary change. The scale bars indicate substitution per site. A list of orthologous protein families is provided in Table S2.

FIG 2

Contrasting phylogenies using Phz proteins and housekeeping enzymes. In some species of Burkholderia, phenazine genes may have been acquired via horizontal gene transfer. Contrasting neighbor-joining phylogenies inferred from concatenated sequences of PhzA/B, PhzE, and PhzG proteins (left) and housekeeping enzymes AtpD, GltB, GyrB, RecA, LepA, PhaC, and TrpB (right). Indels were ignored in the analysis, and the concatenated Phz and housekeeping data sets contained 1,024 and 4,458 characters, respectively. Sequences from P. phenazinium LMG 2247^T were used as an outgroup. Evolutionary distances were estimated using the Jukes-Cantor genetic distance model. The reproducibility of clades was assessed by bootstrap resampling with 1,000 pseudoreplicates, and bootstrap values greater than 60% are indicated by gray circles at the nodes (circle sizes are proportional to bootstrap values). The branch lengths are proportional to the amount of evolutionary change, and the scale bars indicate substitutions per site.

FIG 3

Gene organization of phenazine gene clusters in different species of Burkholderia. Comparison of the phenazine gene cluster of B. lata ATCC 17760 to its counterparts from other Burkholderia and P. phenazinium and to the esmeraldin biosynthesis (esm) locus of Streptomyces antibioticus Tu2706. Core biosynthesis genes are highlighted in red. Homologous modifying genes are indicated by arrows of the same color and connected with shading, whereas unique species-specific genes are shown by open arrows. The arrows with bold outlines indicate known or putative phenazine transport genes. The sizes of genes and intergenic regions are not to scale. Locus tags are shown using a code (e.g., the locus tag for the pcm1 homolog of Burkholderia sp. BDU5 is WS69_11045). The predicted gene functions are summarized in Table S3.

FIG 4

(A) Phenazine intermediates and other secondary metabolites identified in extracts of B. lata ATCC 17760 and (B) comparison of phenazines produced by B. lata and P. synxantha 2-79Z carrying the pUCP26-all plasmid. All phenazine derivatives are shown in blue, while the nonphenazine compounds are shown in green. Intermediates 3 and 8 could not be purified and were identified based on their HPLC-UV-MS profiles.

FIG 5

(A) Genetic organization of the phenazine biosynthesis cluster from B. lata ATCC 17760, isogenic mutants, and pUCP26-based plasmids containing different combinations of phz genes. Predicted genes are shown by colored arrows. DNA fragments cloned in plasmids under the control of lac promoter are indicated by thick lines. Inverted black triangles show insertions of the Tp^r cassette in the genome of B. lata. The right panel depicts structures of phenazines detected in P. synxantha 2-79Z carrying plasmids with phz genes by HPLC-coupled ESI-MS. (B) The proposed role of enzymes encoded by pcm1 and pcm2 in the biosynthesis of dimethyl 4,9-dihydroxy-1,6-phenazinedicarboxylate. (C) The appearance of wild-type B. lata ATCC 17760 and its pcm1 and phzA mutants in King’s medium B.

FIG 6

Comparison of flow cell biofilms formed by B. lata ATCC 17760, its phenazine-deficient mutant B. lata phzA and the phenazine-overproducing derivative B. lata phzA(pBBR1MCS-all). The three stains were inoculated into the BioFlux microfluidic system and allowed to form a biofilm for 48 h. The bright-field images were collected at 20 min intervals with an LS620 digital microscope. The top panel shows the percentage of surface area covered by the growing biofilm, whereas the bottom panel shows representative images of biofilm development over the course of the experiment.

FIG 7

Static biofilms formed by B. lata ATCC 17760 and its phenazine-deficient and complemented derivatives. All strains were suspended in KMB broth at approximately 10⁶ CFU ml⁻¹ and grown in 96-well PVC plates for 48 h at 27°C, at which point the biofilms were stained with crystal violet. Treatments with different letters indicate significant differences as determined by the Kruskal-Wallis rank test followed by Dunn’s multiple comparison test (P < 0.05).