No apparent costs for facultative antibiotic production by the soil bacterium Pseudomonas fluorescens Pf0-1

Abstract

Background: Many soil-inhabiting bacteria are known to produce secondary metabolites that can suppress microorganisms competing for the same resources. The production of antimicrobial compounds is expected to incur fitness costs for the producing bacteria. Such costs form the basis for models on the co-existence of antibiotic-producing and non-antibiotic producing strains. However, so far studies quantifying the costs of antibiotic production by bacteria are scarce. The current study reports on possible costs, for antibiotic production by Pseudomonas fluorescens Pf0-1, a soil bacterium that is induced to produce a broad-spectrum antibiotic when it is confronted with non-related bacterial competitors or supernatants of their cultures.

Methodology and principal findings: We measured the possible cost of antibiotic production for Pseudomonas fluorescens Pf0-1 by monitoring changes in growth rate with and without induction of antibiotic production by supernatant of a bacterial competitor, namely Pedobacter sp.. Experiments were performed in liquid as well as on semi-solid media under nutrient-limited conditions that are expected to most clearly reveal fitness costs. Our results did not reveal any significant costs for production of antibiotics by Pseudomonas fluorescens Pf0-1. Comparison of growth rates of the antibiotic-producing wild-type cells with those of non-antibiotic producing mutants did not reveal costs of antibiotic production either.

Significance: Based on our findings we propose that the facultative production of antibiotics might not be selected to mitigate metabolic costs, but instead might be advantageous because it limits the risk of competitors evolving resistance, or even the risk of competitors feeding on the compounds produced.

Figure 1. Incubation chamber for determination of bacterial growth made of a glass slide with cardboard spacers on both sides.

Between the spacers 3 pre-inoculated water-agar (WA) slices of 1 cm² are placed. A glass coverslip is slightly pressed on top of the agar slices. The sides of the incubation chamber are sealed with parafilm.

Figure 2. Bacterial growth (Optical Density at 600 nm) in nutrient-poor liquid medium in microplates.

: Pf01wt+CFS - P. fluorescens Pf0-1 wild type with 10% cell-free supernatant from Pedobacter sp. V48; Pf01wt+BCFS - P. fluorescens Pf0-1 wild type with 10% boiled cell-free supernatant from Pedobacter sp. V48; Δ3463+CFS – P. fluorescens Pf0-1 mutant Δ3463 with 10% cell-free supernatant from Pedobacter sp. V48 and Δ3463+BCFS – P. fluorescens Pf0-1 mutant Δ3463 with 10% boiled cell-free supernatant from Pedobacter sp. V48. The measurements were performed every 30 min for total period of 8.5 h. Symbols represent means of 3 replicate measurements; error bars represent standard deviations.

Figure 3. Bacterial growth in micro-colonies measured microscopically using Water-Agar incubation chambers.

(A) white squares -wild type P. fluorescens Pf0-1 exposed to 10% cell-free supernatant from Pedobacter sp. V48 and black squares- wild type P. fluorescens Pf0-1 exposed to 10% boiled cell-free supernatant from Pedobacter sp. V48; (B) white squares -wild type P. fluorescens Pf0-1 exposed to 10% cell-free supernatant from Pedobacter sp. V48 and black squares- mutant Δ3463 exposed to 10% cell-free supernatant from Pedobacter sp. V48. Squares represent the means of micro-colony sizes and the error bars represent the standard deviations. Statistical analysis revealed no significant differences (p>0.05) between the growth rates (slopes) of the treatments.

Figure 4. Morphology of bacterial micro-colonies after 7 h of incubation on Water-Agar of (A) wild type P. fluorescens Pf0-1 exposed to 10% cell-free supernatant from Pedobacter sp. V48 (B) wild type P. fluorescens Pf0-1 exposed to 10% boiled cell-free supernatant from Pedobacter sp. V48 and (C) - Mutant Δ3463 exposed to 10% cell-free supernatant from Pedobacter sp. V48.