LuxR- and luxI-type quorum-sensing circuits are prevalent in members of the Populus deltoides microbiome

Abstract

We are interested in the root microbiome of the fast-growing Eastern cottonwood tree, Populus deltoides. There is a large bank of bacterial isolates from P. deltoides, and there are 44 draft genomes of bacterial endophyte and rhizosphere isolates. As a first step in efforts to understand the roles of bacterial communication and plant-bacterial signaling in P. deltoides, we focused on the prevalence of acyl-homoserine lactone (AHL) quorum-sensing-signal production and reception in members of the P. deltoides microbiome. We screened 129 bacterial isolates for AHL production using a broad-spectrum bioassay that responds to many but not all AHLs, and we queried the available genome sequences of microbiome isolates for homologs of AHL synthase and receptor genes. AHL signal production was detected in 40% of 129 strains tested. Positive isolates included members of the Alpha-, Beta-, and Gammaproteobacteria. Members of the luxI family of AHL synthases were identified in 18 of 39 proteobacterial genomes, including genomes of some isolates that tested negative in the bioassay. Members of the luxR family of transcription factors, which includes AHL-responsive factors, were more abundant than luxI homologs. There were 72 in the 39 proteobacterial genomes. Some of the luxR homologs appear to be members of a subfamily of LuxRs that respond to as-yet-unknown plant signals rather than bacterial AHLs. Apparently, there is a substantial capacity for AHL cell-to-cell communication in proteobacteria of the P. deltoides microbiota, and there are also Proteobacteria with LuxR homologs of the type hypothesized to respond to plant signals or cues.

Fig 1

Phylogenetic trees of LuxI-LuxR family members from Populus bacterial isolates (bold lettering) and select Proteobacteria. The scale indicates the number of substitutions per residue, and bootstrap values as the percentage of 100 samples are shown for nodes with values of 50% or greater. (A) Phylogenetic tree of LuxI AHL synthases from members of the Populus bacterial isolates and select Proteobacteria. The subfamily tree of AHL synthases that synthesize atypical QS signals is highlighted in red. OlsB, an ornithine acyltransferase, is included as an outgroup. (B) Phylogenetic tree of LuxR-type receptors from members of the Populus bacterial isolates and select Proteobacteria. The subfamily tree of LuxRs that responds to atypical QS signals is highlighted in red. LysR, a transcriptional regulator containing a helix-turn-helix DNA-binding motif, is included as an outgroup. Detailed information for each LuxI and LuxR homolog is given in Table S2 in the supplemental material.

Fig 2

Phylogenetic tree of likely non-plant-responsive solo LuxR polypeptides from Populus bacterial isolates (bold lettering) and select Proteobacteria. The scale indicates the number of substitutions per residue, and bootstrap values as the percentage of 100 samples are shown for nodes with values of 50% or greater. Each subfamily tree of solo LuxR receptors present in multiple isolates is highlighted in a separate color. The Pseudomonas subfamily PpoR homologs are highlighted in red. The Rhizobium subfamily ExpR homologs are highlighted in turquoise. Rhizobium subfamily LuxR members without a described homolog are highlighted in blue, green, and magenta. LysR, a transcriptional regulator containing a helix-turn-helix DNA-binding motif, is included as an outgroup. Detailed information for each LuxR homolog is given in Table S2 in the supplemental material.

Fig 3

Likely plant-responsive LuxR homologs are present in Populus isolates. (A) Phylogenetic tree of selected probable plant-responsive LuxR family members from Populus bacterial isolates (bold lettering) and selected Proteobacteria. The scale indicates the number of substitutions per residue, and bootstrap values as the percentage of 100 samples are shown for nodes with values of 50% or greater. The subfamily tree of LuxR receptors that respond to a plant compound is highlighted in red. LysR, a transcriptional regulator containing a helix-turn-helix DNA-binding motif, is included as an outgroup. Amino acid sequence information for each LuxR homolog is detailed in Table S2 in the supplemental material. (B) Gene organization map of the plant-responsive luxR and pip genes in Pseudomonas sp. strain GM79 and Rhizobium sp. strains AP16, CF122, CF142, and PD01-076 (similar to nesR in E. meliloti strain 1021). (C) Sequence alignment of putative R-binding sites found upstream from the pip2 and pip3 genes. Site positions in which there is a majority agreement are colored red. The coordinates on the right indicate where the inverted repeat is centered relative to the ATG start site (in bp). A single asterisk indicates that the binding site overlaps the 3′ coding region of the luxR gene, and a double asterisk indicates that the binding site overlaps the 5′ coding region of the luxR gene. The oryR gene is adjacent to only a single downstream pip gene (analogous to pip2); the DNA sequence of the OryR-binding site is shown for comparison.