Rhizonin, the First Mycotoxin Isolated from the Zygomycota, Is Not a Fungal Metabolite but Is Produced by Bacterial Endosymbionts

Abstract

Rhizonin is a hepatotoxic cyclopeptide isolated from cultures of a fungal Rhizopus microsporus strain that grew on moldy ground nuts in Mozambique. Reinvestigation of this fungal strain by a series of experiments unequivocally revealed that this "first mycotoxin from lower fungi" is actually not produced by the fungus. PCR experiments and phylogenetic studies based on 16S rRNA gene sequences revealed that the fungus is associated with bacteria belonging to the genus Burkholderia. By transmission electron microscopy, the bacteria were localized within the fungal cytosol. Toxin production and the presence of the endosymbionts were correlated by curing the fungus with an antibiotic, yielding a nonproducing, symbiont-free phenotype. The final evidence for a bacterial biogenesis of the toxin was obtained by the successful fermentation of the endosymbiotic bacteria in pure culture and isolation of rhizonin A from the broth. This finding is of particular interest since Rhizopus microsporus and related Rhizopus species are frequently used in food preparations such as tempeh and sufu.

Global Distribution and Evolution of a Toxinogenic Burkholderia-Rhizopus Symbiosis

“…In sum, this represents an unparalleled example for a symbiosis in which a fungus harbors bacteria for the production of a virulence factor. In analogy, we found that the first reported "mycotoxins" from lower fungi, the highly toxic cyclopeptides rhizonin A and B (25,28), are also produced by symbiotic bacteria (Burkholderia endofungorum) and not by the fungus (16). While both rhizoxins and rhizonins have been believed to promote zygomycoses (21), there is no indication for toxinproducing endosymbiotic bacteria in clinical isolates (18).…”

“…S1 in the supplemental material). Only one isolate, Burkholderia endofungorum HKI-0456 (isolate B5), also produces the hepatotoxic cyclopeptides rhizonin A (derivative 8) and B (derivative 9) under laboratory conditions (16).…”

Bacterial-Fungal Interactions: Hyphens between Agricultural, Clinical, Environmental, and Food Microbiologists

“…The presence of rhizonin toxins produced by endobacteria inhabiting Rhizopus microsporus is a critical cause of food spoilage due to their hepatotoxicity (296). Similarly, rhizoxin-producing endobacteria present a potential threat in food processing since R. microsporus is used during the fermentative production of tempeh (209,296,326). Bacteria can also cause spoilage on mushroom tissues, although the effect is largely on mushroom yield and cosmetic appeal to the consumer rather than on human health (374).…”

“…Some studies have examined the potential of fungi to assist colonization by human-pathogenic bacteria; for example, spoilage fungi on tomatoes can promote Salmonella colonization, an effect which is believed to be due to their effect on pH (393,394). The presence of rhizonin toxins produced by endobacteria inhabiting Rhizopus microsporus is a critical cause of food spoilage due to their hepatotoxicity (296). Similarly, rhizoxin-producing endobacteria present a potential threat in food processing since R. microsporus is used during the fermentative production of tempeh (209,296,326).…”

Time-Dependent Production of the Bioactive Peptides Endolides A and B and the Polyketide Mariline A from the Sponge-Derived Fungus Stachylidium bicolor 293K04

“…Interestingly, endolides incorporate a 3-(3-furyl)-alanine residue, a rare amino acid previously reported only twice. The first description corresponds to rhizonin A, a peptide described as produced by the zygomycete fungus Rhizopus microsporus [4,5], and later confirmed to be encoded by the symbiotic endobacterium Burkholderia endofungorum [6]. The second report of 3-(3-furyl)-alanine was associated with the cytotoxic bingchamides from Streptomyces bingchenggensis [7], reinforcing the hypothesis of a possible bacterial biosynthetic origin of this rare amino acid.…”