Polyphosphates (polyP) are polymers of orthophosphate residues linked by high-energy phosphoanhydride bonds that are important in all domains of life and function in many different processes, including biofilm development. To study the effect of polyP in archaeal biofilm formation, our previously described Sa. solfataricus polyP (−) strain and a new polyP (−) S. acidocaldarius strain generated in this report were used. These two strains lack the polymer due to the overexpression of their respective exopolyphosphatase gene (ppx). Both strains showed a reduction in biofilm formation, decreased motility on semi-solid plates and a diminished adherence to glass surfaces as seen by DAPI (4′,6-diamidino-2-phenylindole) staining using fluorescence microscopy. Even though arlB (encoding the archaellum subunit) was highly upregulated in S. acidocardarius polyP (−), no archaellated cells were observed. These results suggest that polyP might be involved in the regulation of the expression of archaellum components and their assembly, possibly by affecting energy availability, phosphorylation or other phenomena. This is the first evidence indicating polyP affects biofilm formation and other related processes in archaea.

Acidithiobacillus ferrooxidans resists extremely high concentrations of copper. Strain ATCC 53993 is much more resistant to the metal compared with strain ATCC 23270, possibly due to the presence of a genomic island in the former one. The global response of strain ATCC 53993 to copper was analyzed using iTRAQ (isobaric tag for relative and absolute quantitation) quantitative proteomics. Sixty-seven proteins changed their levels of synthesis in the presence of the metal. On addition of CusCBA efflux system proteins, increased levels of other envelope proteins, such as a putative periplasmic glucan biosynthesis protein (MdoG) involved in the osmoregulated synthesis of glucans and a putative antigen O polymerase (Wzy), were seen in the presence of copper. The expression of A. ferrooxidansmdoG or wzy genes in a copper sensitive Escherichia coli conferred it a higher metal resistance, suggesting the possible role of these components in copper resistance of A. ferrooxidans. Transcriptional levels of genes wzy, rfaE and wzz also increased in strain ATCC 23270 grown in the presence of copper, but not in strain ATCC 53993. Additionally, in the absence of this metal, lipopolysaccharide (LPS) amounts were 3-fold higher in A. ferrooxidans ATCC 53993 compared with strain 23270. Nevertheless, both strains grown in the presence of copper contained similar LPS quantities, suggesting that strain 23270 synthesizes higher amounts of LPS to resist the metal. On the other hand, several porins diminished their levels in the presence of copper. The data presented here point to an essential role for several envelope components in the extreme copper resistance by this industrially important acidophilic bacterium.

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.