Redox-active antibiotics enhance phosphorus bioavailability

Abstract

Microbial production of antibiotics is common, but our understanding of their roles in the environment is limited. In this study, we explore long-standing observations that microbes increase the production of redox-active antibiotics under phosphorus limitation. The availability of phosphorus, a nutrient required by all life on Earth and essential for agriculture, can be controlled by adsorption to and release from iron minerals by means of redox cycling. Using phenazine antibiotic production by pseudomonads as a case study, we show that phenazines are regulated by phosphorus, solubilize phosphorus through reductive dissolution of iron oxides in the lab and field, and increase phosphorus-limited microbial growth. Phenazines are just one of many examples of phosphorus-regulated antibiotics. Our work suggests a widespread but previously unappreciated role for redox-active antibiotics in phosphorus acquisition and cycling.

Fig. 1.. P regulates production of antibiotics in diverse bacteria.

Tree depicts species with experimental evidence for P-limited antibiotic production (pink text) and experimentally confirmed (filled green circle) or likely (open green circle) regulation by phoB/P and phoR. Data are largely from (5) and (6), see Table S1. Metabolite production in B. thailandensis and Serratia ATCC39006 was tested using chemostats (Fig. S1). Example structures for different antibiotics are shown with broad antibiotic type listed above each structure. The common metabolite name and producer are also listed. Producers of each example metabolite are also indicated with numbers around the tree. Tree was built from 241 small subunit rRNA sequences using RaxML (29) and is rooted for display.

Fig. 2.. Phenazine production in different pseudomonads is regulated by phosphate via phoB.

(A) Phenazine production in phosphorus- (blue) or nitrogen- (yellow) limited Pseudomonas chemostats. Phenazines produced are PCA: phenazine-1-carboxylic-acid; PCN: phenazine-1-carboxamide; PYO: pyocyanin. ODs were maintained at ~0.1, the range of ODs across chemostats for each experiment ± standard deviation is listed. (B) Phenazine structures. (C) PhoB is thought to increase phenazine production by binding predicted PHO boxes upstream of phenazine biosynthetic genes and QS genes (19, 21). (D) Phenazine production in wild type Pseudomonas and phoB mutants at μ=0.06. Reported growth rate (μ) is the dilution rate, the two are equivalent at steady state. Nd: not detected, OD: optical density at 500 nm.

Fig. 3.. Phenazines solubilize P and promote P. aeruginosa growth on HFO-P.

(A) Results of reactions between HFO-P and phenazines after 5 hours of anoxic incubation: Fe(II) (ferrozine assay), total soluble iron (ICP-MS), and total soluble phosphorus (ICP-MS). Nd: not detected. Error bars: standard deviations for duplicates. See Fig. S2 for details on P adsorption. (B) Model of reductive dissolution. The reaction of phenazine with Fe(III) is a two electron transfer yielding 2Fe(II) (15). Solubilized P is variable (indicate by n) depending on the extent of P surface coordination as dictated by P concentration, mineral composition, and pH (26). (C) Growth (as a denitrifier) of a P. aeruginosa mutant unable to make phenazines on HFO-P. Additions: 100 μM (reduced phenazine), 7mM (phosphate). Shaded area represents the standard deviation for biological duplicates.

Fig. 4.. Phenazines solubilize P in marine sediments.

(A) Nitric acid extractable iron and phosphorus from sampling sites. Error bars: standard deviations from triplicate digestions. (B-D) Phosphorus solubilization in Catalina Island sediments from Site 1 collected in August (B), and Site 2 collected in August (C) or October (D). Y-axes in (B-D) reflect difference in total soluble phosphorus (ICP-MS) from the initial time point, horizontal lines depict no change. See also Figs. S3, S4. (E) PCA reduction is suppressed in sediments treated with ethanol. (F) PCA reduction is stimulated by organic carbon (10 mM glucose + 10 mM lactate). Sediments in (F) were starved before PCA additions and cannot be compared directly to (E). For (B-F) Plots reflect data from 4 or 5 replicates, outliers (single black dots) are >1.5x interquartile range, p values reflect comparison to the control treatment (as indicated on the figure) at a specific time point. * p<0.05, ** p<0.005, ***, p<0.0001.