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Biofilm engineering through c-di-GMP tuning boosts bioleaching efficiency and arsenic tolerance in Acidithiobacillus ferrooxidans.

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Title: Biofilm engineering through c-di-GMP tuning boosts bioleaching efficiency and arsenic tolerance in Acidithiobacillus ferrooxidans.
Authors: Han, Xi1, Hu, Yidan2, Yue, Yanbo2, Ding, Yuefei1 huyidan@cug.edu.cn, Cao, Bin3, Shi, Liang2,4, Liu, Juan1 juan.liu@pku.edu.cn
Source: Applied & Environmental Microbiology. Mar2026, Vol. 92 Issue 3, p1-17. 17p.
Subjects: Biofilms, Bacterial leaching, Bacteria, Heavy metal toxicology, Synthetic biology, Arsenic poisoning, Second messengers (Biochemistry)
Abstract: Bioleaching offers a sustainable alternative to conventional metallurgy, but its application is limited by low leaching rates, inhibition by heavy metals, and prolonged adaptation. Here, we engineered Acidithiobacillus ferrooxidans, a model bioleaching microorganism ubiquitous in mining environments, by modulating intracellular bis(3′−5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling to enhance biofilm formation, bioleaching efficiency, and arsenic tolerance. Overexpression of diguanylate cyclase genes AFE_1379, AFE_0053, and AFE_1373 produced engineered strains S-222, S-306, and S-651, respectively, with 1.7-, 2.5-, and 5-fold higher intracellular c-di-GMP levels than the control carrying the empty plasmid vector. Under arsenic-free conditions, all engineered strains showed similar growth profiles, but S-306, at intermediate c-di-GMP (306.3 ± 28.1 μg mg-1), formed cytochrome-rich biofilms with low internal resistance and achieved the highest bioleaching efficiency. Under arsenic stress, S-651, at high c-di-GMP (651.4 ± 15.5 μg mg-1), developed polysaccharide-rich biofilms that enhanced arsenic tolerance, scorodite (FeAsO4·2H2O) precipitation, and bioleaching performance. Transcriptomic analysis confirmed these strain-specific gene expression patterns. These findings demonstrate that tuning intracellular c-di-GMP enables A. ferrooxidans to reprogram biofilm matrix composition for extracellular electron uptake and heavy-metal resistance, providing a synthetic biology strategy for environmentally friendly bioleaching and tailings recycling. IMPORTANCE As a model microorganism for bioleaching, Acidithiobacillus ferrooxidans is limited in leaching efficiency by several key constraints, including slow biofilm formation and susceptibility to environmental heavy metals. Although genetic engineering has been widely used to tackle these challenges, conventional strategies typically focus on modifying one single trait at a time, which significantly restricts their industrial applicability. In this study, we present a novel approach that overcomes this limitation through targeted modulation of the global regulatory molecule c-di-GMP. Engineering this upstream signaling pathway allowed for the tunable enhancement of both bioleaching efficiency and heavy metal resistance, providing an integrated strategy to address multiple bottlenecks simultaneously. This work offers a versatile and practical biotechnology route for diverse scenarios to enhance bioleaching performance and environmental adaptability, which may facilitate the utilization of low-grade ores and mining tailings and ultimately contribute to more sustainable and circular metal production. [ABSTRACT FROM AUTHOR]
Copyright of Applied & Environmental Microbiology is the property of American Society for Microbiology and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
Database: Engineering Source
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DbLabel: Engineering Source
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  Label: Title
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  Data: Biofilm engineering through c-di-GMP tuning boosts bioleaching efficiency and arsenic tolerance in Acidithiobacillus ferrooxidans.
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  Data: <searchLink fieldCode="AR" term="%22Han%2C+Xi%22">Han, Xi</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Hu%2C+Yidan%22">Hu, Yidan</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Yue%2C+Yanbo%22">Yue, Yanbo</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Ding%2C+Yuefei%22">Ding, Yuefei</searchLink><relatesTo>1</relatesTo><i> huyidan@cug.edu.cn</i><br /><searchLink fieldCode="AR" term="%22Cao%2C+Bin%22">Cao, Bin</searchLink><relatesTo>3</relatesTo><br /><searchLink fieldCode="AR" term="%22Shi%2C+Liang%22">Shi, Liang</searchLink><relatesTo>2,4</relatesTo><br /><searchLink fieldCode="AR" term="%22Liu%2C+Juan%22">Liu, Juan</searchLink><relatesTo>1</relatesTo><i> juan.liu@pku.edu.cn</i>
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  Data: <searchLink fieldCode="JN" term="%22Applied+%26+Environmental+Microbiology%22">Applied & Environmental Microbiology</searchLink>. Mar2026, Vol. 92 Issue 3, p1-17. 17p.
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  Data: <searchLink fieldCode="DE" term="%22Biofilms%22">Biofilms</searchLink><br /><searchLink fieldCode="DE" term="%22Bacterial+leaching%22">Bacterial leaching</searchLink><br /><searchLink fieldCode="DE" term="%22Bacteria%22">Bacteria</searchLink><br /><searchLink fieldCode="DE" term="%22Heavy+metal+toxicology%22">Heavy metal toxicology</searchLink><br /><searchLink fieldCode="DE" term="%22Synthetic+biology%22">Synthetic biology</searchLink><br /><searchLink fieldCode="DE" term="%22Arsenic+poisoning%22">Arsenic poisoning</searchLink><br /><searchLink fieldCode="DE" term="%22Second+messengers+%28Biochemistry%29%22">Second messengers (Biochemistry)</searchLink>
– Name: Abstract
  Label: Abstract
  Group: Ab
  Data: Bioleaching offers a sustainable alternative to conventional metallurgy, but its application is limited by low leaching rates, inhibition by heavy metals, and prolonged adaptation. Here, we engineered Acidithiobacillus ferrooxidans, a model bioleaching microorganism ubiquitous in mining environments, by modulating intracellular bis(3′−5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling to enhance biofilm formation, bioleaching efficiency, and arsenic tolerance. Overexpression of diguanylate cyclase genes AFE_1379, AFE_0053, and AFE_1373 produced engineered strains S-222, S-306, and S-651, respectively, with 1.7-, 2.5-, and 5-fold higher intracellular c-di-GMP levels than the control carrying the empty plasmid vector. Under arsenic-free conditions, all engineered strains showed similar growth profiles, but S-306, at intermediate c-di-GMP (306.3 ± 28.1 μg mg-1), formed cytochrome-rich biofilms with low internal resistance and achieved the highest bioleaching efficiency. Under arsenic stress, S-651, at high c-di-GMP (651.4 ± 15.5 μg mg-1), developed polysaccharide-rich biofilms that enhanced arsenic tolerance, scorodite (FeAsO4·2H2O) precipitation, and bioleaching performance. Transcriptomic analysis confirmed these strain-specific gene expression patterns. These findings demonstrate that tuning intracellular c-di-GMP enables A. ferrooxidans to reprogram biofilm matrix composition for extracellular electron uptake and heavy-metal resistance, providing a synthetic biology strategy for environmentally friendly bioleaching and tailings recycling. IMPORTANCE As a model microorganism for bioleaching, Acidithiobacillus ferrooxidans is limited in leaching efficiency by several key constraints, including slow biofilm formation and susceptibility to environmental heavy metals. Although genetic engineering has been widely used to tackle these challenges, conventional strategies typically focus on modifying one single trait at a time, which significantly restricts their industrial applicability. In this study, we present a novel approach that overcomes this limitation through targeted modulation of the global regulatory molecule c-di-GMP. Engineering this upstream signaling pathway allowed for the tunable enhancement of both bioleaching efficiency and heavy metal resistance, providing an integrated strategy to address multiple bottlenecks simultaneously. This work offers a versatile and practical biotechnology route for diverse scenarios to enhance bioleaching performance and environmental adaptability, which may facilitate the utilization of low-grade ores and mining tailings and ultimately contribute to more sustainable and circular metal production. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
  Label:
  Group: Ab
  Data: <i>Copyright of Applied & Environmental Microbiology is the property of American Society for Microbiology and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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RecordInfo BibRecord:
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      – Type: doi
        Value: 10.1128/aem.02288-25
    Languages:
      – Code: eng
        Text: English
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        PageCount: 17
        StartPage: 1
    Subjects:
      – SubjectFull: Biofilms
        Type: general
      – SubjectFull: Bacterial leaching
        Type: general
      – SubjectFull: Bacteria
        Type: general
      – SubjectFull: Heavy metal toxicology
        Type: general
      – SubjectFull: Synthetic biology
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      – SubjectFull: Arsenic poisoning
        Type: general
      – SubjectFull: Second messengers (Biochemistry)
        Type: general
    Titles:
      – TitleFull: Biofilm engineering through c-di-GMP tuning boosts bioleaching efficiency and arsenic tolerance in Acidithiobacillus ferrooxidans.
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            NameFull: Han, Xi
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            NameFull: Hu, Yidan
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            NameFull: Yue, Yanbo
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            – D: 01
              M: 03
              Text: Mar2026
              Type: published
              Y: 2026
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            – TitleFull: Applied & Environmental Microbiology
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