Acidophiles play a dominant role in driving elemental cycling in natural acid mine drainage (AMD) habitats and exhibit important application value in bioleaching and bioremediation. Acidity is an inevitable environmental stress and a key factor that affects the survival of acidophiles in their acidified natural habitats; however, the regulatory strategies applied by acidophilic bacteria to withstand low pH are unclear. We identified the significance of the ferric uptake regulator (Fur) in acidophiles adapting to acidic environments and discovered that Fur is ubiquitous as well as highly conserved in acidophilic bacteria. Mutagenesis of the fur gene of Acidithiobacillus caldus, a prototypical acidophilic sulfur-oxidizing bacterium found in AMD, revealed that Fur is required for the acid resistance of this acidophilic bacterium. Phenotypic characterization, transcriptome sequencing (RNA-seq), mutagenesis, and biochemical assays indicated that the Acidithiobacillus caldus ferric uptake regulator (AcFur) is involved in extreme acid resistance by regulating the expression of several key genes of certain cellular activities, such as iron transport, biofilm formation, sulfur metabolism, chemotaxis, and flagellar biosynthesis. Finally, a Fur-dependent acid resistance regulatory strategy in A. caldus was proposed to illustrate the ecological behavior of acidophilic bacteria under low pH. This study provides new insights into the adaptation strategies of acidophiles to AMD ecosystems and will promote the design and development of engineered biological systems for the environmental adaptation of acidophiles. IMPORTANCE This study advances our understanding of the acid tolerance mechanism of A. caldus, identifies the key fur gene responsible for acid resistance, and elucidates the correlation between fur and acid resistance, thus contributing to an understanding of the ecological behavior of acidophilic bacteria. These findings provide new insights into the acid resistance process in Acidithiobacillus species, thereby promoting the study of the environmental adaptation of acidophilic bacteria and the design of engineered biological systems.

Acidithiobacillaceae , an important family of acidophilic and chemoautotrophic sulfur or iron oxidizers, participate in geobiochemical circulation of the elements and drive the release of heavy metals in mining associated habitats. Because of their environmental adaptability and energy metabolic systems, Acidithiobacillus spp. have become the dominant bacteria used in bioleaching for heavy metal recovery. Flagella-driven motility is associated with bacterial chemotaxis and bacterial responses to environmental stimuli. However, little is known about how the flagellum of Acidithiobacillus spp. is regulated and how the flagellum affects the growth of these chemoautotrophic bacteria. In this study, we analyzed the flagellar gene clusters in Acidithiobacillus strains and uncovered the close relationship between flagella and the sulfur-oxidizing systems (Sox system). The σ 28 gene ( rpoF ) knockout and overexpression strains of Acidithiobacillus caldus were constructed. Scanning electron microscopy shows that A. caldus Δ rpoF cells lacked flagella, indicating the essential role of RpoF in regulating flagella synthesis in these chemoautotrophic bacteria. Motility analysis suggests that the deletion of rpoF resulted in the reduction of swarming capability, while this capability was enhanced in the rpoF overexpression strain. Both static cultivation and low concentration of energy substrates (elemental sulfur or tetrathionate) led to weak growth of A. caldus Δ rpoF cells. The deletion of rpoF promoted bacterial attachment to the surface of elemental sulfur in static cultivation. The absence of RpoF caused an obvious change in transcription profile, including genes in flagellar cluster and those involved in biofilm formation. These results provide an understanding on the regulation of flagellar hierarchy and the flagellar function in these sulfur or iron oxidizers.

Acid resistance of acidophiles is the result of long-term co-evolution and natural selection of acidophiles and their natural habitats, and formed a relatively optimal acid-resistance network in acidophiles. The acid tolerance network of acidophiles could be classified into active and passive mechanisms. The active mechanisms mainly include the proton efflux and consumption systems, generation of reversed transmembrane electrical potential, and adjustment of cell membrane composition; the passive mechanisms mainly include the DNA and protein repair systems, chemotaxis and cell motility, and quorum sensing system. The maintenance of pH homeostasis is a cell-wide physiological process that adopt differently adjustment strategies, deployment modules, and integration network depending on the cell’s own potential and its habitat environments. However, acidophiles exhibit obvious strategies and modules similarities on acid resistance because of the long-term evolution. Therefore, a comprehensive understanding of acid tolerance network of acidophiles would be helpful for the intelligent manufacturing and industrial application of acidophiles.

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