Competition Sensing Changes Antibiotic Production in Streptomyces

Abstract

One of the most important ways that bacteria compete for resources and space is by producing antibiotics that inhibit competitors. Because antibiotic production is costly, the biosynthetic gene clusters coordinating their synthesis are under strict regulatory control and often require "elicitors" to induce expression, including cues from competing strains. Although these cues are common, they are not produced by all competitors, and so the phenotypes causing induction remain unknown. By studying interactions between 24 antibiotic-producing strains of streptomycetes, we show that strains commonly inhibit each other's growth and that this occurs more frequently if strains are closely related. Next, we show that antibiotic production is more likely to be induced by cues from strains that are closely related or that share secondary metabolite biosynthetic gene clusters (BGCs). Unexpectedly, antibiotic production is less likely to be induced by competitors that inhibit the growth of a focal strain, indicating that cell damage is not a general cue for induction. In addition to induction, antibiotic production often decreases in the presence of a competitor, although this response was not associated with genetic relatedness or overlap in BGCs. Finally, we show that resource limitation increases the chance that antibiotic production declines during competition. Our results reveal the importance of social cues and resource availability in the dynamics of interference competition in streptomycetes.IMPORTANCE Bacteria secrete antibiotics to inhibit their competitors, but the presence of competitors can determine whether these toxins are produced. Here, we study the role of the competitive and resource environment on antibiotic production in Streptomyces, bacteria renowned for their production of antibiotics. We show that Streptomyces cells are more likely to produce antibiotics when grown with competitors that are closely related or that share biosynthetic pathways for secondary metabolites, but not when they are threatened by competitor's toxins, in contrast to predictions of the competition sensing hypothesis. Streptomyces cells also often reduce their output of antibiotics when grown with competitors, especially under nutrient limitation. Our findings highlight that interactions between the social and resource environments strongly regulate antibiotic production in these medicinally important bacteria.

Keywords: Streptomyces; antibiotic production; interference competition; microbial ecology; social microbiology.

FIG 1

Schematic of constitutive and facultative inhibition assays. Focal strains (orange) were tested for their capacity to inhibit each target strain (gray) inoculated on top of the focal colony in a soft agar overlay. Inhibition was detected as a zone of clearance surrounding the colony. All 24 strains were tested as both focal and target strains, leading to 576 possible assays for constitutive antibiotic production. For the facultative assays, a second colony was inoculated 1 cm away, designated the competitor, that could interact with the focal strain through diffusible molecules. All 24 strains were tested each as the focal, competitor, and target strain, resulting in 24 × 24 × 24 = 13,824 assays. All assays were conducted under both high- and low-resource conditions. Comparison of the ability of the focal strain to inhibit the target in the constitutive and facultative assays revealed whether antibiotic production was induced, suppressed, or unchanged.

FIG 2

Constitutive antagonism. (A) Inhibition matrix sorted by multilocus sequence typing (MLST) relatedness. Triangles indicate whether a target strain showed growth (white) or was inhibited (black) by the focal strain. Each square is divided into two triangles: the upper triangle shows the results under high-resource conditions, while the lower triangle shows the results under low-resource conditions. Self-inhibition is denoted in blue. Missing data due to inconsistent results are shown in gray. Panels B to D show results of assays conducted at high resource levels. (B) Correlation between inhibition phenotype dissimilarity (Euclidian distance determined by calculating the dissimilarity between focal strain inhibition phenotypes) and phylogenetic distance (Mantel test, P < 0.001, r = 0.27, n = 552) or (C) biosynthetic gene cluster (BGC) distance (Mantel test, P < 0.001, r = 0.43, n = 552). Dots are semitransparent: darker areas represent overlapping data points. (D) Logistic regression between the probability of inhibition and phylogenetic and biosynthetic gene cluster (BGC) distance (P_{phylogenetic distance} < 0.001, P_{BGC distance} = 0.046, McFadden R² = 0.02, n = 536).

FIG 3

Altered antagonism during coculture under high-resource conditions. (A) Interaction heat map showing changes to target strain inhibition when a focal strain is grown in coculture with a competitor. Each square is divided into two triangles: the upper triangle shows induction in red, while the lower triangle shows suppression in blue. Gray triangles indicate that either induction or suppression was not possible for this focal strain, due to the result in the constitutive assay. (A strain that does not inhibit any targets cannot be suppressed, while a strain that inhibits all targets cannot be induced.) (B) Gray bars indicate the number of target strains inhibited by the focal strain when grown alone. Black dots indicate the net number of target strains inhibited by the same focal strain if it was induced and/or suppressed during coculture with one of the 24 possible competitors. Dots showing the same number of inhibited target strains as the gray bar indicate that a competitor strain causes an equal level of induction and suppression against targets, resulting in no net change. (C) Number of competitors that induce or suppress each focal strain. Cases where suppression is not possible due to the absence of constitutive inhibition are denoted as “NA.”

FIG 4

Induction during coculture. (A) The probability that a focal strain is induced is lower when the competitor is antagonistic to the focal strain under both high-resource (logistic regression, P < 0.001, McFadden R² = 0.06, n = 354) and low-resource (logistic regression, P < 0.001, McFadden R² = 0.12, n = 419) conditions. (B) Logistic regressions between the probability of induction and phylogenetic distance under high-resource (black line) and low-resource (dashed line) conditions (P < 0.001, McFadden R² = 0.02, n = 487 and P = 0.205, McFadden R² = 0.00292, n = 445, respectively) or (C) logistic regressions between the probability of induction and BGC distance under high-resource (black line) and low-resource (dashed line) conditions (P < 0.001, McFadden R² = 0.04, n = 487 and P = 0.174, McFadden R² = 0.003, n = 445, respectively). Shaded areas indicate SE.

FIG 5

Altered antagonism during coculture under low-resource conditions. (A) Interaction heat map showing change in target strain inhibition when a focal strain is grown in coculture with a competitor. Each square is divided into two triangles: the upper triangle shows induction in red, while the lower triangle shows suppression in blue. Gray triangles indicate that either induction or suppression was not possible for this focal strain, due to the result in the constitutive assay. (A strain that does not inhibit any targets cannot be suppressed, while a strain that inhibits all targets cannot be induced.) (B) Gray bars indicate the number of strains inhibited by the focal strain when grown alone. Black dots indicate the net number of target strains inhibited by the same focal strain if it was induced and/or suppressed during coculture with one of the 24 possible competitors. Dots showing the same number of inhibited target strains as the gray bar indicate that a competitor strain causes an equal level of induction and suppression against targets, resulting in no net change. (C) Number of competitors that induce or suppress each focal strain. Cases where suppression is not possible due to the absence of constitutive inhibition are denoted as “NA.”

FIG 6

Constitutive and facultative inhibition under high- and low-resource conditions. Shown are comparisons of the total amount of inhibition, change in inhibition due to competition, and induction and suppression found under low- and high-resource conditions.