Effect of Endosymbiotic Bacteria on Fungal Resistance Toward Heavy Metals

Most studies on metal removal or tolerance by fungi or bacteria focus on single isolates, without taking into consideration that some fungi in nature may be colonized by endobacteria. To address this knowledge gap, we investigated the tolerance and removal of diverse metals with two fungal species: Linnemannia elongata containing Burkholderia-related endobacteria and Benniella erionia containing Mollicute-related endobacteria. Isogenic lines of both species were generated with antibiotic treatments to remove their respective endobacteria. Experiments involved comparing the isogenic lines and wild type fungi in relation to the minimum inhibitory concentration for the metals, the fungal ability to remove these different metals via atomic adsorption spectroscopy, and the interaction of the metals with specific functional groups of the fungi and fungi-bacteria to determine the role of the bacteria via attenuated total reflection fourier transformed infrared (ATR-FTIR). Finally, we determined the influence of different metal concentrations, associated with moderate and high fungal growth inhibition, on the presence of the endobacteria inside the fungal mycelium via quantitative real-time PCR. Results showed that the presence of the endosymbiont increased B. erionia resistance to Mn²⁺ and increased the removal of Fe²⁺ compared to isogenic lines. The absence of the endosymbiont in L. elongata increased the fungal resistance toward Fe²⁺ and improved the removal of Fe²⁺. Furthermore, when the bacterial endosymbiont was present in L. elongata, a decrease in the fungal resistance to Ca²⁺, Fe²⁺, and Cr⁶⁺was noticeable. In the ATR-FTIR analysis, we determined that C-H and C = O were the major functional groups affected by the presence of Cu²⁺, Mn²⁺, and Fe²⁺ for L. elongata and in the presence of Cu²⁺ and Ca²⁺ for B. eronia. It is noteworthy that the highest concentration of Pb²⁺ led to the loss of endobacteria in both L. elongata and B. eronia, while the other metals generally increased the concentration of endosymbionts inside the fungal mycelium. From these results, we concluded that bacterial endosymbionts of fungi can play a fundamental role in fungal resistance to metals. This study provides the first step toward a greater understanding of symbiotic interactions between bacteria and fungi in relation to metal tolerance and remediation.