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

## TL;DR

This paper explores how the extremophile Acidithiobacillus caldus uses CsrA and rpoS to regulate its adaptation to harsh environments, improving its survival and bioleaching efficiency.

## Contribution

The study reveals a novel post-transcriptional regulatory mechanism involving CsrA and rpoS in Acidithiobacillus caldus, enhancing its adaptation to extreme conditions.

## Key findings

- CsrA overexpression in A. caldus increases acid tolerance, flagella length, and extracellular secretion.
- CsrA binds to rpoS mRNA, suppressing its translation and regulating biofilm composition and motility-related genes.
- Overexpression of csrA improves bioleaching efficiency by 20.81%.

## Abstract

Acidithiobacillus caldus 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 csrA expression recombinant strain was constructed in A. caldus 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 rpoS leader mRNA. CsrA suppresses rpoS mRNA translation by competing with ribosomes for binding sites, thereby negatively regulating rpoS 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 (flgC, 07035, motD, 15040), which also implies the global regulatory role of CsrA. In summary, a potential overall post-transcriptional regulatory mechanism based on CsrA and rpoS by extremophile A. caldus was proposed. Finally, the efficiency of bioleaching application by csrA overexpression strain was improved by 20.81%.