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Gene expression levels of Acidithiobacillus ferrooxidans with varying intracellular c-di-GMP concentrations during bioleaching

ABSTRACT: Bioleaching, a sustainable and environmentally friendly emerging technology, relies on Acidithiobacillus ferrooxidans as a model microorganism for extracting metals from ores. This study investigated how intracellular cyclic diguanylate (c-di-GMP) concentrations regulate biofilm composition and bioleaching performance in A. ferrooxidans. By constructing strains with varying c-di-GMP levels, it was found that moderate c-di-GMP concentrations enhance bioleaching efficiency by upregulating genes associated with extracellular electron transfer (e.g., omcB and cyc2) in biofilms, a mechanism linked to optimized iron oxidation pathways. In contrast, high c-di-GMP levels improve arsenic resistance by promoting exopolysaccharide (EPS) synthesis in biofilms, likely through activation of pel-like operons (e.g., pelD), which strengthen biofilm structural integrity under toxic conditions. These findings reveal a dual regulatory role of c-di-GMP: prioritizing leaching efficiency via electron transport in low-arsenic environments and enhancing stress resistance through EPS production in high-arsenic scenarios. This study provides a theoretical foundation for tailoring A. ferrooxidans to diverse industrial bioleaching environments, leveraging its metabolic versatility and biofilm adaptability. The integration of synthetic biology approaches (e.g., genetic engineering of c-di-GMP pathways) further highlights potential applications for optimizing bioleaching strains.

ORGANISM(S): Acidithiobacillus ferrooxidans

PROVIDER: GSE294636 | GEO | 2026/02/11

REPOSITORIES: GEO

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Project description:CsrA is a post-transcriptional regulator that controls biofilm formation, virulence, carbon metabolism, and motility, among other phenotypes in bacteria. Although CsrA has been extensively studied in γ-proteobacteria and firmicute, it is unknown which cellular processes are targeted for regulation in δ-proteobacteria. In this work, we constructed and characterized the ΔcsrA mutant strain in Geobacter sulfurreducens to determine the involvement of the CsrA protein in the regulation of biofilm and extracellular electron transfer. The ΔcsrA mutant strain grows on acetate-fumarate and reduces soluble Fe(III) similarly to the wild type strain, but has higher rates of insoluble Fe(III) reduction than the wild type. Quantification and characterization of biofilms by violet staining and confocal laser scanning microscopy showed that the ΔcsrA strain produces up to twice as much biofilm as the wild type strain, containing more than 95% viable cells. Transcriptome analysis by RNA-seq shows that in ΔcsrA biofilms developed on an inert support, 244 (103 upregulated and 141 downregulated) genes changed their expression, including those related to extracellular electron transfer, exopolysaccharide synthesis, c-di-GMP synthesis and degradation. To validate the transcriptome data, RT-qPCR confirmed the differential expression of several selected genes in the ΔcsrA strain. Current production in microbial fuel cells was tested and the ΔcsrA strain was found to produce 45-50% more current than the wild type. To verify the genes that changed expression in the graphite electrodes in the ΔcsrA strain, a transcriptome analysis was performed. 181 genes changed their expression in the ΔcsrA biofilms of which 113 genes showed differentially expression only in MFC and 68 genes changed their expression as well as transcriptome from biofilms grown on inert support. In silico analysis of the 5'-UTR regions revealed that 76 genes that changed expression in the RNA-seq analysis have a consensus sequence for CsrA binding. This is the first report describing the involvement of CsrA in the regulation of extracellular electron transfer and biofilm in a member of the γ-proteobacteria.

Project description:Bacterial lifestyles are dictated by the conditions encountered during colonization of a specific ecological niche or host. A wide range of environmental stimuli triggers sensory modules, that modify activity of proteins, or/and initiate a transfer of information in complex regulatory networks, which modulate gene expression, and therefore adaptation. The transition between planktonic and biofilm growth is dependent on the homeostasis of the intracellular second messenger c-di-GMP. High c-di-GMP levels driven by diguanylate cyclase (DGC) activity favor biofilm while low levels maintained by phosphodiesterase (PDE) enzymes encourage planktonic style or biofilm dispersal. In Pseudomonas aeruginosa over 40 PDE/DGC are involved in c-di-GMP homeostasis, including 16 dual enzymes possessing both canonical DGC and PDE motifs, i.e. GGDEF and EAL, respectively. It has been previously reported that the deletion of the PDE/DGC dual enzyme PA0285, one of 5 c-di-GMP-related enzymes conserved across all Pseudomonas species, impacts biofilms and causes elevated c-di-GMP levels in P. aeruginosa. Here we confirm that this role is conserved in various P. aeruginosa (PAO1, PA14 and PAK) and Pseudomonas putida strains. Deletion of PA0285 has the highest impact during the early stage of surface colonization, and RNA-seq analysis in biofilm context indicates that it may be associated with upregulation of cupA fimbrial genes. We demonstrate that the C-terminal portion of PA0285 encompassing the GGDEF and EAL domains binds the respective GTP (GGDEF) and c-di-GMP (EAL) substrates but only exhibits PDE activity in vitro. Both GGDEF and EAL domains are important for PA0285 PDE activity in vivo as suggested by site-directed mutants in each of the key motifs. Complementation of the PA0285 mutant strain with the C-terminal GGDEF/EAL portion in trans is not as effective as with the full-length gene suggesting that the transmembrane region and PAS domains contained in PA0285’s N-terminal portion influence its PDE activity in vivo. We speculate that the transmembrane portion or/and PAS domains of PA0285 impede its PDE activity in vivo through modulating the conformation of the enzyme, as shown for P. aeruginosa RbdA, in response to an unknown stimulus.

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Gene expression levels of Acidithiobacillus ferrooxidans with varying intracellular c-di-GMP concentrations during bioleaching

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