Surface-active antibiotic production as a multifunctional adaptation for postfire microorganisms

Abstract

Wildfires affect soils in multiple ways, leading to numerous challenges for colonizing microorganisms. Although it is thought that fire-adapted microorganisms lie at the forefront of postfire ecosystem recovery, the specific strategies that these organisms use to thrive in burned soils remain largely unknown. Through bioactivity screening of bacterial isolates from burned soils, we discovered that several Paraburkholderia spp. isolates produced a set of unusual rhamnolipid surfactants with a natural methyl ester modification. These rhamnolipid methyl esters (RLMEs) exhibited enhanced antimicrobial activity against other postfire microbial isolates, including pyrophilous Pyronema fungi and Amycolatopsis bacteria, compared to the typical rhamnolipids made by organisms such as Pseudomonas spp. RLMEs also showed enhanced surfactant properties and facilitated bacterial motility on agar surfaces. In vitro assays further demonstrated that RLMEs improved aqueous solubilization of polycyclic aromatic hydrocarbons, which are potential carbon sources found in char. Identification of the rhamnolipid biosynthesis genes in the postfire isolate, Paraburkholderia kirstenboschensis str. F3, led to the discovery of rhlM, whose gene product is responsible for the unique methylation of rhamnolipid substrates. RhlM is the first characterized bacterial representative of a large class of integral membrane methyltransferases that are widespread in bacteria. These results indicate multiple roles for RLMEs in the postfire lifestyle of Paraburkholderia isolates, including enhanced dispersal, solubilization of potential nutrients, and inhibition of competitors. Our findings shed new light on the chemical adaptations that bacteria employ to navigate, grow, and outcompete other soil community members in postfire environments.

Keywords: antibiotics; fire; interspecies interactions; motility; surfactants.

Figure 1

(A) Ecologically based natural product discovery framework; bacterial isolates from postfire soils were cultivated in parallel on a rich medium and PyOM-containing medium; solid cultures were screened using an agar plug assay for antifungal activity against P. omphalodes; (B) preliminary antifungal screening results for select postfire bacterial isolates F3, C1, C2, F6, and D6-W; bacteria were cultivated on PyOM agar and a rich medium (International Streptomyces Project 2, ISP2 agar) and assayed against P. omphalodes P1672; zones of inhibition from select Paraburkholderia-produced metabolites were larger from PyOM cultures compared to ISP2 cultures; isolate F6 cultivated on either medium did not inhibit P. omphalodes, while isolate D6-W inhibited P. omphalodes when cultivated on ISP2, but not on PyOM.

Figure 2

Structural elucidation of novel rhamnolipid methyl esters; (A) proposed chemical structure for the P. kirstenboschensis F3-produced RLME A; dashed arrows indicate theoretical molecular ion fragments that produce m/z values observed (obs) in the experimental data shown in (B) along with theoretically calculated values (calc); (B) HR-MS/MS fragmentation spectra obtained using collision energy of 15 eV; (C) rhamnolipid methyl esters produced by P. kirstenboschensis F3 with details for each analog, and the known Rhamnolipid 1 produced by P. aeruginosa; structural differences are highlighted in red; theoretical m/z values and ppm errors were calculated using the Barrow group online calculator tool; chemical formulas represent neutral species.

Figure 3

Rhamnolipid methyl ester biosynthesis genes are clustered in P. kirstenboschensis F3; (A) genotypes of WT and knockout mutants; heatmap of production of RLME and biosynthetic intermediates of P. kirstenboschensis F3 wild type and knockout mutants, observed using LC–MS; relative abundance (RA) of possible rhl pathway intermediates found in P. kirstenboschensis F3 knockout strains and wild type (WT), by extracted ion chromatogram peak area and normalized by RA within each strain (slate blue, more abundant; tan, less abundant); (B) genes in the RLME BGC with their amino acid (aa) length, Pfam annotation, and proposed function; the newly identified rhlM gene encodes for a putative Class VI integral membrane methyltransferase; (C) proposed pathway for biosynthesis of di-RLME A in P. kirstenboschensis F3; dRL, di-rhamnolipid; dRLME, dirhamnolipid methyl ester; ACP, acyl carrier protein; dTDP, deoxythymidine diphosphate; mRL, mono-rhamnolipid.

Figure 4

(A) P. kirstenboschensis F3 WT- or rhl mutant-conditioned plugs tested against P. omphalodes; images are representative of four biological replicates per strain and at least three independent experimental replicates; structures to the right represent the major rhl pathway product for each strain; the RLME methyl group is highlighted in red; scale bar is 1 cm; (B) clearance zone diameters for P. kirstenboschensis F3 WT, rhl mutant strains, and genetic complement strains tested against P. omphalodes; data are representative of four replicates per strain; different letters indicate a statistically significant difference as determined by a one-way ANOVA and post hoc Tukey’s test (P < .05); dashed line indicates mean wildtype measurement; (C) inhibition of P. omphalodes using different concentrations of purified RLME A from P. kirstenboschensis F3 or Rhamnolipids (Di-Rhamnolipid dominant mixture) from P. aeruginosa (Sigma-Aldrich); compounds were solubilized and diluted in methanol and 15 μl of each solution was applied in the central well; images are representative of three technical replicates; (D) quantification of the inhibition zone diameters from tested RLME A and Rhamnolipids mixture; points represent the mean of three replicates for each condition, and error bars represent standard deviation.

Figure 5

(A) Motility phenotypes (top) and surfactant zone production (bottom) for P. kirstenboschensis F3 WT and rhl mutants; images are representative of at least five biological replicates per strain and at least three independent experimental replicates; scale bar is 1 cm; (B) solubilization of naphthalene (left), phenanthrene (center), and benzo[a]pyrene (right) in water supplemented with either 500 mg/l RLME A (1) from P. kirstenboschensis F3 or 500 mg/l Rhamnolipids (RLs, Di-Rhamnolipid dominant mixture, Sigma-Aldrich) from P. aeruginosa; different letters indicate a statistically significant difference as determined by a one-way ANOVA and post hoc Tukey’s test (P < .05).

Figure 6

Race tube assay for inhibition of P. omphalodes 1672 on pyrolyzed organic matter (PyOM); (A) schematic for race tube assay; P. omphalodes was inoculated at one end of the race tube opposite from either PBS (negative control), P. kirstenboschensis F3 (F3) wildtype (WT), or F3 ΔrhlA; arrows indicate direction of P. omphalodes growth; images were acquired 16.5 cm from the P. omphalodes inoculation point and taken from above; scale bar is 1 mm; (B) P. omphalodes growth over time, measured by distance from point of inoculation; data points are mean values of either six biological replicates (1672 vs. F3 WT and F3 ΔrhlA), or five biological replicates (1672 alone); error bars represent standard deviation; statistical significance was measured by Welch’s t-test between two conditions at a single timepoint; gray asterisks indicate statistically significant differences between 1672 alone and when inoculated across from F3 WT; red asterisks indicate statistically significant differences between 1672 inoculated across F3 WT and when inoculated across F3 ΔrhlA; ^*P ≤ .05,  ^**P ≤ .01.

Figure 7

(A) AlphaFold model for RhlM (gray) alone, aligned with M. acetivorans methyltransferase (PDB: 4a2n, cyan), and aligned with T. castaneum ICMT (PDB: 5vg9, pink); SAH is shown in green; the membrane is indicated by two solid horizontal lines; (B) surface representation of the Rank 1 pose from Maestro ligand docking in RhlM, using an applied positional constraint for substrate carboxylate O and SAM methyl C; docked pose was verified by molecular dynamics using Desmond; surface is colored by molecular lipophilicity potential (gold = lipophilic, cyan = hydrophilic); (C) cross-section of RhlM with substrate and cofactor bound; the upper hydrophobic region provides access for the lipid region of the RL substrate, while the lower region accommodates the carboxylate terminus, optimally positioning the carboxylate for methyl transfer from the SAM cofactor; measured C-O distance is shown in magenta.

Figure 8

(A) Sequence similarity network (SSN) for ICMT protein family; nodes are colored by domain: red, Bacteria; blue, Eukarya; yellow, Archaea; gray, unclassified; cyan V-shape indicates P. kirstenboschensis F3 RhlM, magenta diamonds indicate that a crystal structure is available; clusters containing three or fewer nodes are omitted for clarity; (B) enlarged view of Cluster 14 from the SSN; cyan-colored nodes indicate that the RhlM homolog sequence is found within a rhamnolipid biosynthetic context, and that the strain is of the family Burkholderiaceae; (C) species tree of the BSL clade, showing presence of the rhamnolipid BGC (red circle) and RhlM (cyan circle) within genomes of each species identified using BLAST; the outgroup (Bacillus subtilis str. 168) was manually removed to facilitate visualization.