Post-Transcriptional Regulatory Mechanism Based on CsrA and rpoS in Extremophile Sulfur Oxidizer Acidithiobacillus caldus

<i>Acidithiobacillus caldus</i> is perpetually exposed to multiple extreme environmental stresses. CsrA, functioning as a post-transcriptional regulator of physiological metabolism, acts as a differential modulator, facilitating more economical and efficient adaptation to extreme environments. The <i>csrA</i> expression recombinant strain was constructed in <i>A. caldus</i> MTH-04 by conjugative transfer technology pJD215. Physiological characterization revealed enhanced acid tolerance, significantly elongated flagella, elevated extracellular secretion, and altered biofilm composition. Notably, intracellular concentrations of free glutamate and aspartate increased to 24.18 mg/L and 16.07 mg/L, respectively. The secondary structure of CsrA protein was determined in vitro through circular dichroism spectroscopy and size-exclusion chromatography. Electrophoretic Mobility Shift Assay (EMSA) successfully demonstrated in vitro binding activity of CsrA to the <i>rpoS</i> leader mRNA. CsrA suppresses <i>rpoS</i> mRNA translation by competing with ribosomes for binding sites, thereby negatively regulating <i>rpoS</i> expression. Critical binding sites were further validated through site-directed mutagenesis. Through EMSA, RT-qPCR and the translation reporter system, it was also found that CsrA has a dual regulatory function for nearby flagella- and motility-related gene clusters (<i>flgC, 07035, motD, 15040</i>), which also implies the global regulatory role of CsrA. In summary, a potential overall post-transcriptional regulatory mechanism based on CsrA and <i>rpoS</i> by extremophile <i>A. caldus</i> was proposed. Finally, the efficiency of bioleaching application by <i>csrA</i> overexpression strain was improved by 20.81%.