Bacterial Associates of a Desert Specialist Fungus-Growing Ant Antagonize Competitors with a Nocamycin Analog

Abstract

Fungus-growing ants are defended by antibioticproducing bacterial symbionts in the genus Pseudonocardia. Nutrients provisioned by the ants support these symbionts but also invite colonization and competition from other bacteria. As an arena for chemically mediated bacterial competition, this niche offers a window into ecological antibiotic function with welldefined competing organisms. From multiple colonies of the desert specialist ant Trachymyrmex smithi, we isolated Amycolatopsis bacteria that inhibit the growth of Pseudonocardia symbionts under laboratory conditions. Using bioassay-guided fractionation, we discovered a novel analog of the antibiotic nocamycin that is responsible for this antagonism. We identified the biosynthetic gene cluster for this antibiotic, which has a suite of oxidative enzymes consistent with this molecule's more extensive oxidative tailoring relative to similar tetramic acid antibiotics. High genetic similarity to globally distributed soil Amycolatopsis isolates suggest that this ant-derived Amycolatopsis strain may be an opportunistic soil strain whose antibiotic production allows for competition in this specialized niche. This nocamycin analog adds to the catalog of novel bioactive molecules isolated from bacterial associates of fungus-growing ants, and its activity against ant symbionts represents, to our knowledge, the first putative ecological function for the widely distributed enoyl tetramic acid family of antibiotics.

The insect microbiome is a vast source of bioactive small molecules

“…The novel analog nocamycin V ( 69 ) was identified as the antibacterial compound. 211 Nocamycin V ( 69 ) belongs to the enoyl tetramic acid family of antibiotics, differing from nocamycin I by the addition of a hydroxyl group in the cyclic ketal moiety. Nocamycins have a PKS–NRPS hybrid scaffold, which undergoes oxidative tailoring.…”

“…Conversely, Amycolatopsis strains are not sensitive to 69 , suggesting that nocamycin V is likely mediating bacterial competition in this ecological context, despite not exhibiting any antifungal activity against the specific fungal pathogen Escovopsis . 211…”

“…It also has four putative cytochromes P450 in total, including NmvG and NmvO found in nocamycin I. 211,212 This family is comprised of compounds that inhibit bacterial RNA polymerase, thus displaying activity against Gram-positive bacteria. 213 In agar diffusion assays, 10 mg of 69 produced signicant zones of inhibition in the culture of symbiotic Pseudonocardia strains collected not only from T. smithi, but also from other Trachymyrmex species and other fungus-growing ants.…”

“…Conversely, Amycolatopsis strains are not sensitive to 69, suggesting that nocamycin V is likely mediating bacterial competition in this ecological context, despite not exhibiting any antifungal activity against the specic fungal pathogen Escovopsis. 211 The known antibiotic EC0-0501 (70) was also produced by Amycolatopsis isolated from desert-adapted fungus-growing ants. 214 This glycosidic polyene bears an aminohydroxycyclopentenone moiety and it is a potent antibacterial against some Gram-positive strains such as methicillinresistant S. aureus, vancomycin-resistant Enterococci faecalis and B. subtilis (MIC = 2, 8 and 1 mg mL −1 , respectively).…”

Symbiosis, dysbiosis and the impact of horizontal exchange on bacterial microbiomes in higher fungus-gardening ants

“…The most heavily studied microbial symbionts within the attine ant system are bacteria that are thought to have defensive functions, especially Actinobacteria (Pseudonocardia, Streptomyces and Amycolatopsis) and Burkholderia [26][27][28][29] . Such bacteria are suggested to aid in the suppression of predatory fungi that consume the fungus garden by producing secondary metabolites that function as antibiotics 28,30 . Other bacterial associates of the symbiosis appear to have a nutritional function.…”