Peptide-based quorum sensing systems in Paenibacillus polymyxa

(A) Phylogenetic tree of 234 AloR homologs found in 14 P. polymyxa strains. Colored segments in the tree circumference define clades of AloR proteins corresponding to the systems found in P. polymyxa ATCC 842 (labeled as arrows). Systems immediately followed by a Spo0E-like protein are indicated. Tree was constructed using IQ-Tree (55, 56, 57) with 1,000 iterations, and ultrafast bootstrap support values are presented for major branches. Tree visualization was done with iTOL (58). Alo1/8 refers to a system duplicated in P. polymyxa ATCC 842 (where it is represented as Alo1 and Alo8). (B) Frequency and distribution of Alo systems in P. polymyxa strains. The number of copies per Alo system found within P. polymyxa strains is shown using colored squares. The absence of an Alo system in a specific strain is marked by a white square. (A) Phylogenetic tree of the Alo systems shown on the vertical axis is derived from the tree in the panel (A). Phylogenetic tree of the strains shown on the horizontal axis is based on the GyrA protein, with GyrA from B. subtilis 168 as an out-group.

(A) Phr peptide fragments detected in the growth media of B. subtilis 168. Peptide fragments identified by MS are underlined, with the cumulative number of spectra detected in all tested repeats indicated (see the Materials and Methods section). Known mature communication peptides are in red (26, 45). The two most abundantly identified fragments are presented for each protein by an underline. The mature peptides which were not directly detected are inferred from the C-terminal sequence of the full peptides, presumably after processing. (B) AloP peptide fragments detected by MS. Same as (A) for AloP peptide fragments detected in the growth media of P. polymyxa ATCC 842.

(A) Volcano plot depicting P. polymyxa gene expression after incubation with 5 μM of the peptide SHGRGG for 10 min, as compared to control conditions in which no peptide was added. X-axis, log₂ fold expression change (adjusted using the lfcShrink function in the DESeq2 R package (62); see the Materials and Methods section). Y-axis, −log₁₀ P-value. Average of three independent replicates; each dot represents a single gene. Dots appearing in colors are genes that passed the threshold for statistical significance of differential expression (see the Materials and Methods section). Red dots correspond to genes encoded by Alo systems. (B) Differentially expressed genes as appears in panel (A). Fold change represents the average of the three independent replicates. P-adjusted is the P-value after correction for multiple hypothesis testing (see the Materials and Methods section). (C) RNA-seq coverage of the Alo13-Alo14 locus (Scaffold: PPTDRAFT_AFOX01000049_1.49), in control conditions (green) or 10 min after addition of 5 μM of the SHGRGG peptide (black). RNA-seq coverage is in log scale and was normalized by the number of uniquely mapped reads in each condition. Representative of three independent replicates. (D) RNA-seq coverage of the Alo13-Alo14 locus in control conditions (green) or 10 min after addition of 5 μM of a scrambled version (GRGSGH) of the AloP13 peptide. RNA-seq coverage is in log scale and normalized as in the panel (C). Representative of three independent replicates. (E) Schematic representation of the genetic construct used in B. subtilis to verify the activity of AloR13. The start codon of the aloP13 gene (dashed blue arrow) was mutated to inactivate it. GFP was placed as a reporter gene instead of the Spo0E ORF. (F) GFP fluorescence upon addition of the AloP13 peptide SHGRGG in varying concentrations, or the scrambled mature peptide GRGSGH in 5 μM (“scrambled”), or no peptide added. Dashed lines represent GFP fluorescence measured without the addition of IPTG.