Some archaea from the genus Sulfolobus are important for bioleaching of copper, where metal resistant microorganisms are required. Biofilm generation is one of the ways microorganisms cope with some stimuli in nature, including heavy metals. The response to external factors, particularly in the biofilm form of life, is still underexplored in archaea. To explore how model thermoacidophilic archaeon Saccharolobus solfataricus faces copper stress during this lifestyle, changes in biofilms were studied using crystal violet staining, confocal fluorescence microscopy, and qPCR approaches. It was found that biofilm formation reached a maximum at 0.5 mM Cu, before starting to decrease at higher metal concentrations. The morphology of biofilms at 0.5 mM Cu was observed to be different, displaying lower thickness, different sugar patterns, and higher amounts of cells compared to standard growing conditions. Furthermore, copA, which is responsive to intracellular Cu concentration, was downregulated in biofilm cells when compared with planktonic cells exposed to the same metal concentration. The latest results suggests that cells in biofilms are less exposed to Cu than those in planktonic culture. In a PolyP-deficient strain, Cu was not able to induce biofilm formation at 0.5 mM. In summary, the findings reported here suggest that the biofilm form of life confers S. solfataricus advantages to face stress caused by Cu.Biofilm formation remains a relatively unexplored topic in archaeal research. Therefore, this knowledge in model organisms such as S. solfataricus, and how they use it to face stress, could be of great importance to engineer organisms with improved capabilities to be applied in biotechnological processes, such as bioleaching of metals.

Polyphosphates are biopolymers composed of phosphate monomers linked by high-energy phosphoanhydride bonds. They are present across all life domains, serving as a source of energy, metal chelators, and playing a crucial role in stress defense. In Escherichia coli, polyphosphates also function as inorganic molecular chaperones. The present study aims to investigate whether polyphosphate serves a similar chaperone function in archaea, using Saccharolobus solfataricus as a model organism. To this end, polyphosphate was extracted and quantified, the ADP/ATP ratio was determined, insoluble protein extracts were analyzed at different time points after copper exposure, and qPCR was performed to measure the expression of stress-related genes. PolyP was extracted after exposing the archaeon S. solfataricus to different copper concentrations. We determined that polyP degradation is directly correlated with metal concentration. At the minimum inhibitory concentration (MIC) of 2 mM Cu²⁺, polyP degradation stabilized 2 h after exposure and showed no recovery even after 24 h. The ADP/ATP ratio was measured and showed differences in the presence or absence of polyP. The analysis of proteins precipitated under copper stress showed a higher proportion of insoluble proteins at an elevated metal concentration. On the other hand, increased protein precipitation was detected in the absence of polyP. Gene expression analysis via qPCR was conducted to assess the expression of genes involved in chaperone and chaperonin production, copper resistance, oxidative stress response, and phosphate metabolism under prolonged copper exposure, both in the presence and absence of polyP. The results indicated an upregulation of all the chaperonins measured in the presence of polyP. Interestingly, just some of these genes were upregulated in polyP’s absence. Despite copper stress, there was no upregulation of superoxide dismutase in our conditions. These results highlight the role of polyP in the copper stress response in S. solfataricus, particularly to prevent protein precipitation, likely due to its function as an inorganic chaperone. Additionally, the observed protein precipitation could be attributable to interactions between copper and some amino acids on the protein structures rather than oxidative stress induced by copper exposure, as previously described in E. coli. Our present findings provide new insights into the protective role of polyP as an inorganic chaperone in S. solfataricus and emphasize its importance in maintaining cellular homeostasis under metal stress conditions.

Conserved Histidine Alpha-helical Domain (CHAD) proteins attached to the surface of polyphosphate (PolyP) have been studied in some bacteria and one archaeon. However, the activity of CHAD proteins is unknown beyond their interaction with PolyP granules. By using bioinformatic analysis, we report that several species of the biomining acidophilic bacteria contain orthologs of CHAD proteins with high sequence identity. Furthermore, the gene coding for the CHAD protein is in the same genetic context of the enzyme polyphosphate kinase (PPK), which is in charge of PolyP synthesis. Particularly, the group of ppk and CHAD genes is highly conserved. Metallosphaera sedula and other acidophilic archaea used in biomining also contain CHAD proteins. These archaea show high levels of identity in genes coding for a cluster having the same organization. Amongst these genes are chad and ppx. In general, both biomining bacteria and archaea contain high PolyP levels and are highly resistant to heavy metals. Therefore, the presence of this conserved genetic organization suggests a high relevance for their metabolism. It has been formerly reported that a crystallized CHAD protein contains a copper-binding site. Based on this previous knowledge, in the present report, it was determined that all analyzed CHAD proteins are very conserved at their structural level. In addition, it was found that the lack of YgiF, an Escherichia coli CHAD-containing protein, decreases copper resistance in this bacterium. This phenotype was not only complemented by transforming E. coli with YgiF but also by expressing CHAD from Acidithiobacillus ferrooxidans in it. Interestingly, the strains in which the possible copper-binding sites were mutated were also more metal sensitive. Based on these results, we propose that CHAD proteins are involved in copper resistance in microorganisms. These findings are very interesting and may eventually improve biomining operations in the future.