RESUMO
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.
RESUMO
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.
RESUMO
The woody nature of grapevine (Vitis vinifera L.) has hindered the development of efficient gene editing strategies to improve this species. The lack of highly efficient gene transfer techniques, which, furthermore, are applied in multicellular explants such as somatic embryos, are additional technical handicaps to gene editing in the vine. The inclusion of geminivirus-based replicons in regular T-DNA vectors can enhance the expression of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) elements, thus enabling the use of these multicellular explants as starting materials. In this study, we used Bean yellow dwarf virus (BeYDV)-derived replicon vectors to express the key components of CRISPR/Cas9 system in vivo and evaluate their editing capability in individuals derived from Agrobacterium-mediated gene transfer experiments of 'Thompson Seedless' somatic embryos. Preliminary assays using a BeYDV-derived vector for green fluorescent protein reporter gene expression demonstrated marker visualization in embryos for up to 33 days post-infiltration. A universal BeYDV-based vector (pGMV-U) was assembled to produce all CRISPR/Cas9 components with up to four independent guide RNA (gRNA) expression cassettes. With a focus on fungal tolerance, we used gRNA pairs to address considerably large deletions of putative grape susceptibility genes, including AUXIN INDUCED IN ROOT CULTURE 12 (VviAIR12), SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER 4 (VviSWEET4), LESION INITIATION 2 (VviLIN2), and DIMERIZATION PARTNER-E2F-LIKE 1 (VviDEL1). The editing functionality of gRNA pairs in pGMV-U was evaluated by grapevine leaf agroinfiltration assays, thus enabling longer-term embryo transformations. These experiments allowed for the establishment of greenhouse individuals exhibiting a double-cut edited status for all targeted genes under different allele-editing conditions. After approximately 18 months, the edited grapevine plants were preliminary evaluated regarding its resistance to Erysiphe necator and Botrytis cinerea. Assays have shown that a transgene-free VviDEL1 double-cut edited line exhibits over 90% reduction in symptoms triggered by powdery mildew infection. These results point to the use of geminivirus-based replicons for gene editing in grapevine and other relevant fruit species.